Sample fixation and stabilisation

ABSTRACT

It has surprisingly been discovered that it is possible to stabilise biomolecules such as RNA, DNA and proteins in biological samples such as cells, tissues, biopsies and blood using deep eutectic solvents (DES). It has also been discovered that DES mixtures can be used to fix and preserve cell morphology in biological samples such as tissue blocks, cancer biopsies and whole blood. This invention describes methods to stabilise and preserve biomolecules, whole cells, tissues, blood and biological samples using DES mixtures.

PRIORITY APPLICATION INFORMATION

This application is a divisional application of U.S. application Ser.No. 15/608,300 filed May 30, 2017, which is a divisional application ofU.S. application Ser. No. 14/193,680, filed Feb. 28, 2014, which claimspriority to United Kingdom Patent Application GB 1303666.0 filed Mar. 1,2013, all of which are incorporated by reference in their entiretyherein.

FIELD OF THE INVENTION

The present invention relates to the stabilisation of biomolecules, inparticular RNA, including methods for stabilising RNA, compositions andkits for stabilising RNA, and stabilised RNA-containing compositions. Italso relates to the fixation and stabilisation of cells, tissues, blood,exosomes and other biological samples.

BACKGROUND OF THE INVENTION

The extraction of intact biomolecules from a biological sample is anessential part of many laboratory and clinical diagnostic procedures.The instability of biomolecules such as nucleic acids, proteins,carbohydrates and lipids is well known and their integrity depends on alarge number of parameters such as the physiological condition of thesample prior to removal from its original environment, how quickly thesample was removed from its source, the rate of sample cooling, samplestorage temperature and the biomolecule purification method. It is wellknown that the treatment of the biological sample before and duringbiomolecule purification can lead to very important changes in theintactness and integrity of the sample analyte. For example it is wellknown that RNA in particular is an extremely labile molecule thatbecomes completely and irreversibly damaged within minutes if it is nothandled correctly. Although RNA is perhaps one of the more labilebiomolecules, proteins including post-translational modifications,lipids, small molecules of less than 2000 daltons that are essential tometabolic analysis and DNA can also be subject to substantialdegradative processes.

Although endogenous cellular enzymes are responsible for the majority ofdegradative processes, the analyte will always tend to hydrolysespontaneously during storage or processing and this process is largelydependent on the storage conditions such as temperature, water content,pH, light and stability and molecular weight of the analyte molecule butmay also be dependent on the quality of the reagents used.

One of the most common and simple approaches for successful storage isto reduce the temperature of the biological sample. Generally samplesare stored at temperatures below room temperature (20-24° C.); proteinsolutions at 4° C. or −20° C., nucleic acids in freezers at −20° C. or−80° C., in dry-ice or in liquid nitrogen. Anti-microbial agents such assodium azide may be added to control microbial growth.

It is well known that RNA is particularly sensitive to degradation byenzymes, spontaneous hydrolysis, divalent metal cation catalysedhydrolysis, alkali sensitivity and cross-linking in FFPE (formalin-fixedparaffin-embedded) samples. Many metal solutions such as lead, magnesiumand manganese are very destructive to RNA and are indeed essential fornot only ribozyme but also nuclease activity such as DNase I, mung beannuclease and S1 nuclease. Iron (2+) has been implicated in the oxidationof nucleobases as part of the Fenton reaction leading to translationallyimpaired rRNA and mRNA (Honda et al., (2005) J. Biol. Chem. 280,20978-20986), for example the conversion of guanine to 8-oxo-guanine.Other possible catalytic roles of metal ions in enzymatic andnonenzymatic cleavages of phosphodiester bonds have been reviewed(Yarus, M. (1993) FASEB J. 7, 31-39). Indeed chelators such as EDTA andEGTA are frequently added to RNA or RNA lysis solutions for the purposeof reducing RNA degradation by removing metal ions. Ribonucleases(“RNases”) are a large group of ubiquitous enzymes associated with manysources including microbes, human skin, dust and the content of cellsand tissues. They are also readily released from intra-cellular vesiclesduring freeze-thawing. Certain tissues including the pancreas are knownto be particularly rich in RNase A. RNase A is one of the most stableenzymes known, readily regaining its enzymatic activity following, forexample, chaotropic salt denaturation making it extremely difficult todestroy. A high concentration of chaotrope such as guanidine (4-6M) isrequired to destroy RNase activity (Thompson. J. and Gillespie. D. AnalBiochem. (1987) 163:281-91).

There are several methods for inhibiting the activity of RNases such asusing; (i) ribonuclease peptide inhibitors (“RNasin”) an expensivereagent only available in small amounts and specific for RNase A, B andC, (ii) reducing agents such as dithiothreitol and β-mercaptoethanolwhich disrupt disulphide bonds in the RNase enzyme, but the effect islimited and temporary as well as being toxic and volatile, (iii)proteases such as proteinase K to digest the RNases, but the transportof proteinases in kits and their generally slow action allows theanalyte biomolecules to degrade, (iv) reducing the temperature to belowthe enzyme's active temperature; commonly tissue and cellular samplesare stored at −80° C. or in liquid nitrogen, (v) anti-RNase antibodies,(vi) precipitation of the cellular proteins including RNases, DNA andRNA using solvents such as acetone or kosmotropic salts such as ammoniumsulphate, a commercialised preparation of ammonium sulphate is known asRNAlater™ (Sigma-Aldrich, USA; LifeTechnologies, USA; Qiagen, Germany),(vii) detergents to stabilise nucleic acids in whole blood such as thatfound in the PAXgene™ DNA and RNA extraction kit (PreAnalytix, Germany),(viii) chaotropic salts, (ix) alcohols such as those found in thePAXgene™ Tissue stabilisation reagent (PreAnalytix, Germany). A range ofsuch chaotropic mixtures are set out in RNA Isolation and Analysis,Editor. Jones (1994) Chapter 2.

The primary goal of sample storage is to minimise any changes to theanalyte biomolecule that may be introduced as a result of thepre-analytical procedure and sample purification so that the analyticalresult resembles as closely as possible the original in vivo complexityand diversity of the biomolecules, thereby improving assay accuracy,sensitivity and specificity. Whilst there are various methods andproducts that are available to reduce pre-analytical variation, allsuffer from various drawbacks making their use problematic orsub-optimal. Procedures that are effective at stabilising one class ofbiomolecules are often ineffective at stabilising others so that thetechnician is obliged to choose a specialised reagent and procedure foreach biomolecule analyte. For example the PAXgene™ system (PreAnalytix)(U.S. Pat. Nos. 6,602,718 and 6,617,170) can be used for nucleic acidsbut not proteins and requires lengthy purification steps with multiplewash buffers, whilst cocktails of protease inhibitors help to protectproteins from degradation but not nucleic acids. The PAXgene™ tissuestabilisation kit requires two separate treatments of the tissue andinvolves toxic and flammable chemicals. RNAlater™ treatment of tissuesreduces their utility for immunohistochemistry, histology and increasestissue hardness without fully protecting the RNA, nevertheless it hasbeen adopted as the gold standard for RNA preservation.

It is not always possible to purify RNA at the time or site where thesample is extracted, for example a biopsy from a hospital operatingtheatre or a blood sample from a doctor's office. In these cases, thesample must be very carefully stored prior to RNA extraction, whichmight be carried out within as little as 30 minutes but would morecommonly occur only after several hours or days following processing bythe hospital pathology laboratory. Often the time and temperature of thepre-analytical step is poorly recorded leading to ambiguous knowledge ofthe quality of the sample. As a consequence, it has been necessary todevelop separate sample storage conditions for each type of tissue andfinal use of the RNA. As already stated this generally involves usingeither a dedicated stabilisation solution such as RNAlater or PAXgene orimmediately freezing the sample in liquid nitrogen. At least in the caseof the PAXgene stabilisation reagent, incomplete removal of thestabiliser will negatively impact RNA yields during purification(PAXgene Blood RNA Kit Handbook, June 2005).

Tissue storage may be effected by tissue fixation using a fixative.‘Fixation’ refers to increasing the mechanical strength, hardening,preserving and increasing stability of the treated biological samplesuch as fresh cells, biopsy or tissue, and maintains the sample in astate as similar as possible to that of the original fresh sample insitu, in its natural state. Fixation is commonly used in pathology,histology, histochemistry, cytochemistry, anatomical studies andstudying cells, and generally precedes additional steps such as storage,embedding, staining, immunohistochemistry and/or immunocytochemistry.The process of fixation ideally inhibits enzymes such as nucleases andproteases, stops microbial growth on the sample and maintains both grosstissue morphology as well as cellular ultrastructure such as golgi,nucleus, endoplasmic reticulum, mitochondria, lysosomes and cytoplasmicmembranes. As one example, the preservation of the correct cellmorphology is important for a pathologist to diagnose the presence, typeand grade of cancer in a patient, but in order to do this correctly thesample must also be capable of becoming correctly stained or labelledwith antibodies for immunohistochemistry. Commonly the sample is treatedwith a 1-5% aqueous buffered solution of formalin (formaldehyde)paraformaldehyde or glutaldehyde for 1-24 hours at room temperature inorder to allow cross linking of proteins and other cellular componentsand then, following tissue sectioning, stained with Haematoxylin andEosin stain (H&E). Although glutaraldehyde can also be used its rate ofpenetration into the tissue is slower than with formaldehyde (whichpenetrates at approximately 1 mm per hour when 18-20 volumes are addedrelative to the tissue volume). Whilst RNA can also be preserved in thismanner, in general it becomes highly degraded during or after formalinfixation making gene expression analysis highly problematic andartifactual. One specific problem is that the RNA analyte becomescross-linked with other biomolecules such as proteins so that theysubsequently need to be released prior to analysis, this process isgenerally very harsh requiring extended periods at elevated temperatureswhich leads to significant RNA degradation. Another problem of fixationis maintaining soluble analytes such as RNA and proteins in the cell sothey can be integrated by for example, in situ hybridization orimmunohistochemistry. Yet another problem with formalin fixation inparticular is that the covalent modification of the cellular proteinsresults in the loss of antigenic immunorecognition which can renderimmunohistochemistry techniques difficult or impossible depending on theantibody. As one further example, formalin fixed tissues are routinelyembedded in paraffin wax to allow the tissue block to be thinly slicedand examined microscopically (FFPE). Other common tissue fixationmethods involve using methanol, ethanol or acetone that result inprotein precipitation rather than cross-linking. Methods to fix tissues,maintain good RNA quality whilst allowing immunohistochemical stainingare particularly needed. It is well known that commonly used fixativessuch as formaldehyde, paraformaldehyde, gluataldehyde and methanol arehighly toxic potential carcinogens, whilst ethanol and acetone arehighly flammable. The ideal fixative should work on a wide variety oftissues including neural, lymphoid and fatty, preserve large pieces oftissues, and be compatible with immunohistochemical, histochemical andin situ hybridisation and other specialised techniques. It should alsobe compatible with automated tissue fixation procedures. A review withdetailed protocols has been published by Bancroft (2008) ‘Theory andPractice of Histological Techniques’ and by Stanta (2011) ‘Guidelinesfor Molecular Analysis in Archive Tissues’ whilst representativeexamples of fixed and stained tissues can be found in Ross and Pawlina(2011) ‘Histology A Text and Atlas’.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides the use of a deep eutecticsolvent to inhibit the degradation of a biomolecule.

It has surprisingly been discovered that it is possible to stabilisebiomolecules such as RNA, DNA and proteins in biological samples withdeep eutectic solvents (DES). It has also been discovered that DESmixtures can be used to fix and preserve cell morphology in biologicalsamples such as tissue blocks, cancer biopsies and whole blood. Thisinvention describes methods to stabilise and preserve biomolecules,whole cells, tissues and biological samples using DES mixtures. Thesample or tissue may comprise a solid tissue or non-solid tissue.

We have surprisingly found that many DES mixtures are capable ofstabilising and preserving RNA, other biomolecules such as protein andDNA, cells and tissue structures. Using individual DES components alonein aqueous solution such as Choline chloride (6M) or Urea (5M) does notprovide for either RNA stabilisation or cell fixation, only when thecomponents are combined in a DES such as Choline chloride:Urea do theypreserve and stabilise. (Table 1). We have found for example only, thatthe mixture of Choline chloride with Trifluoroacetamide is particularlyeffective at stabilising RNA in cells whilst maintaining cellmorphology. We have also found that the mixture of Choline chloride withsorbitol is particularly effective at stabilising RNA in whole tissues.However an extremely large number of other combinations and ratios ofDES components are useful to practice this invention.

The separation of components of mixtures by natural ionic liquids andnatural DES's has been reported (WO 2011/155829) but the effect of DESon the stabilisation of biomolecules or cells has not been reported.Indeed in US 2009/0117628A1 it has been shown that enzymatic reactionsmay be carried out in a DES, and that <<a variety of enzymes reactionsare active in a variety of DES's>>, indicating that nucleases andproteases and other catabolic enzymes would be equally active.Surprisingly we have found that RNA and other biomolecules arestabilised in DES mixtures and cell morphology is fixed demonstratingthat the cytoskeleton is also stabilised.

DES's are mixtures of two or more components that when combined togetherhave a eutectic point, which is the temperature of solidification orfreezing (Fp). The eutectic point of the combined components isgenerally much lower than either of the components individually or atany other ratio of mixing and occurs at a single temperature withoutseparation of the individual components on solidification. Theproperties of DES's have been described in A. P. Abbott, G. Capper, D.L. Davies, R. K. Rasheed, V. Tambyrajah, Chem. Commun. 7 (2003) 70-71,A. P. Abbott, D. Boothby, G. Capper, D. L. Davies, R. K. Rasheed, J. Am.Chem. Soc. 126 (2004) 9142-9147, [7] G. Imperato, E. Eibler, J.Niedermaier, B. Konig, Chem. Commun. 9 (2005) 1170-1172, S. Gore, S.Baskaran, B. Koenig, Green Chem. 13 (2011) 1009-1013, J. T. Gorke, F.Srienc, R. J. Kazlauskas, Chem. Commun. 10 (2008) 1235-1237, A. P.Abbott, J. Collins, I. Dalrymple, R. C. Harris, R. Mistry, F. Qiu, J.Scheirer, W. R. Wise, Aust. J. Chem. 62 (2009) 341-347, Y. H. Choi, J.van Spronsen, Y. Dai, M. Verberne, F. Hollmann, I. W. C. E. Arends, G.J. Witkamp, R. Verpoorte, Plant Physiol. 156 (2011) 1701-1705, andreviewed by Zhang Q, De Oliveira Vigier K, Royer S, Jerome F. Chem SocRev. 2012 Nov. 7; 41(21):7108-46.

Industrially DES's have been used for electrochemical plating, miningapplications and drill lubricants (US2009/0247432), industrial enzymeapplications (US2009/0117628A1), preparation of inorganic compounds (D.Freudenmann, S. Wolf, M. Wolff and C. Feldmann, Angew. Chem., Int. Ed.,(2011), 50, 11050-11060) or organic compounds (S. Gore, S. Baskaran andB. Koenig, Green Chem., (2011), 13, 1009-1013), biological extractions(WO 2011/155829), in electrochemistry as electrolytes for dye-sensitizedsolar cells and metal electropolishing (H.-R. Jhong, D. Shan-Hill, C.-C.Wan, Y.-Y. Wang and T.-C. Wei, Electrochem. Commun., (2009), 11,209-211), electrodeposition (E. Gomez, P. Cojocaru, L. Magagnin and E.Valles, J. Electroanalytical Chem., (2011), 658, 18-24), purification ofbiodiesel (K. Shahbaz, F. S. Mjalli, M. A. Hashim and I. M. AlNashef,Energy Fuels, (2011), 25, 2671-2678), solubilisation of drugs (H. G.Morrison, C. C. Sun and S. Neervannan, Int. J. Pharm., (2009), 378,136-139), solubilisation of metal oxides (A. P. Abbott, D. Boothby, G.Capper, D. L. Davies and R. K. Rasheed, J. Am. Chem. Soc., (2004), 126,9142-9147) and solubilisation of CO2 (X. Li, M. Hou, B. Han, X. Wang andL. Zou, J. Chem. Eng. Data, (2008), 53, 548-550). Van Spronsen et al.sets out the use of DES's of natural origin such as various combinationsof amino-acids, sugars and carboxylic acids for extraction inWO2011/155829 but does not mention their use for stabilisation or celland tissue fixation. The DES's described are for extraction purposesrather than stabilisation and fixation.

DES's are not considered to be ionic liquids because (i) they are notentirely composed of ionic species and (ii) they can also be obtainedfrom non-ionic species, (iii) they are mixtures and not compounds. Ascompared to the traditional ionic liquids, DES's derived from, forexample, Choline chloride have several advantages such as (1) low cost,(2) chemically inert to water, (3) easy to prepare by simply mixing twoor more components, (4) most are biodegradable and non-toxic, (5) lowvolatility even when heated and (6) non-flammable. All DES's are liquidsbelow 150° C. and many are liquid between room-temperature and 70° C.,with a few notable examples that are liquid below 0° C.

Frequently a DES is obtained by mixing a quaternary ammonium halide saltwith a metal salt such as ZnCl2 or a hydrogen bond donor such as Urea,that has the ability to form a complex with the halide anion of thequaternary ammonium salt. However it has been found that there are alsoexceptions such as when there is no halide anion present in the DES asin Betaine:Trifluoroacetamide. As used herein, the term “betaine” refersto N,N,N-trimethylglycine, unless otherwise specified.

The deep eutectic solvents discussed herein may include a componenthaving a quaternary ammonium group.

The component having a quaternary ammonium group may be a zwitterioniccomponent further comprising a carboxylate group. A preferredzwitterionic component is betaine (N,N,N-trimethylglycine).

Alternatively, the component including a quaternary ammonium group maybe in the form of a salt with an appropriate counterion. The counterionis suitably a halide, and is preferably chloride, bromide or iodide,most preferably chloride. Other suitable counterions include nitrate,tetrafluoroborate, and the like. When the component is in the form of asalt, the counterion preferably does not comprise a carboxylate group.

In a further aspect, the present invention provides a method forstabilising RNA in an RNA-containing sample, which method comprisescontacting the sample with a DES containing mixture to form a stabilisedRNA-containing composition.

In a further aspect, the present invention provides a method forstabilising DNA in a DNA-containing sample, which method comprisescontacting the sample with a DES containing mixture to form a stabilisedDNA-containing composition.

In a further aspect, the present invention provides a method forstabilising proteins including phosphoproteins in a peptide-containingsample, which method comprises contacting the sample with a DEScontaining mixture to form a stabilised protein-containing composition.

In a further aspect, the present invention provides a method for fixinga cell's native morphology, and stabilising RNA, DNA and/or proteins, ina bacterial, fungal, animal or plant sample, such as E. coli, Yeast,Nematode, Drosophila, Zebra fish, Mouse, Rat, Arabidopsis thaliana,Rice, Wheat, Maize, Tobacco and/or Potato.

In a further aspect, the present invention provides a method for fixingand stabilising a cell's native morphology, in a cell containing sample,such as an organ, tissue, biopsy, circulating tumour cell (CTC), bloodsample, plasma sample, serum sample, tissue culture cells, saliva,urine, cerebral spinal fluid, medical sample, egg, embryo, or adulttissue, or FFPE sample, which method comprises contacting the cellcontaining sample with a DES containing mixture to form a fixative andstabiliser of cell structure and morphology compatible with cellcounting, CTC detection, immunohistochemistry, histochemistry, stainingand colouration, flow cytometry, mass spectrometry, in situhybridisation, laser capture microdissection and molecular analyses offor example RNA, DNA and/or proteins.

In a further aspect, the present invention provides a method for fixingand stabilising a cell's native morphology, in a cell containing sample,whereby the protein content and protein cellular localisation can bedetermined by use of a colourant, an antibody or an aptamer, whichmethod comprises contacting the cell containing sample with a DEScontaining mixture to form a fixative and stabiliser of cell structureand morphology and at the same time or subsequently the sample iscontacted with a colourant, an antibody or an aptamer.

In a further aspect, the present invention provides a method for fixingand stabilising a cell's native morphology in a cell containing sample,whereby the RNA content and its cellular localisation can be determinedby use of a labelled or unlabelled probe such as an oligonucleotide, aprimer, a PNA, a LNA, a DNA, a bDNA or RNA sequence, a natural orsynthetic complementary nucleic acid sequence, which method comprisescontacting the cell containing sample with a DES containing mixture toform a fixative and stabiliser of cell structure and morphology and atthe same time or subsequently contacting the sample with a natural orsynthetic hybridising sequence for molecular analysis.

In a further aspect, the present invention provides a kit forstabilising RNA, DNA and/or protein in a biological sample, which kitcomprises at least one container containing a DES mixture. The kitfurther comprising instructions for contacting the sample with a DESmixture whereby the RNA, DNA and/or protein in the biological sample isstabilised against degradation.

In a further aspect, the present invention provides a kit forstabilising cell structure in a biological sample, which kit comprisesat least one container containing a DES mixture. The kit furthercomprising instructions for contacting the sample with a DES mixture,whereby the DES mixture serves as a fixative and/or stabiliser of thecell structure for microscope and/or histological studies such asstaining and colouration, ISH and/or IHC.

In a further aspect, the present invention provides a kit for fixingcell and tissue structures and morphology in a biological sample, whichkit comprises at least one container containing a DES fixative and/orstabilising mixture and optionally wherein the container is permeableand of maximum height of 10 mm into which the sample is immersed andfixed and/or stabilised. The walls of the permeable container can be asieve or grating or provided with slits allowing initial passage andaccess of the DES mixture to the cell or tissue sample, and/orsubsequent contact with liquid paraffin.

In a further aspect, the present invention provides use of a combinationof a DES mixture with an additive to function as a fixative andstabiliser of biomolecules including RNA, DNA and/or proteins.

In a further aspect, the present invention provides a stabilisedRNA-containing composition, which comprises RNA and a DES mixture.

In a further aspect, the present invention provides a stabilisedDNA-containing composition, which comprises DNA and a DES mixture.

In a further aspect, the present invention provides a stabilisedprotein-containing composition, which comprises protein and a DESmixture.

In a further aspect, the present invention provides use of a DES mixtureas a stabiliser of cell structure and/or tissue histology and/ormorphology, and a stabiliser of RNA, DNA and/or proteins.

In a further aspect, the present invention provides use of a DES mixtureas a stabiliser of cell structure and/or tissue histology and/ormorphology, for subsequent embedding in paraffin and microtomesectioning, and optionally staining, in situ hybridisation and/orimmunohistochemistry.

In a further aspect, the present invention provides use of a DES mixtureas a stabiliser of cell structure and/or tissue histology and/ormorphology, for subsequent frozen cryostat sectioning, and optionallystaining, in situ hybridisation and/or immunohistochemistry.

In a further aspect, the present invention provides use of a DES mixtureas a stabiliser of cell structure and/or tissue histology andmorphology, for subsequent laser capture microdissection (LCM) andoptionally recovery of protein, DNA and RNA.

In a further aspect, the present invention provides use of a DES mixtureas a stabiliser of biomolecules and/or cells in plasma, serum and bloodfor subsequent diagnostic analysis including circulating tumour cellsand/or white blood cells and/or whole blood including cell capture,counting and/or typing, staining and/or colouration, mass spectrometry,in situ hybridisation and/or immunohistochemistry. Optionally recoveryof protein, DNA and RNA can be carried out.

In a further aspect the present invention provides use of a DES mixtureas a stabiliser of exosomes, other types of vesicles, microvesicles,sub-cellular bodies and particles such as micelles, liposomes,endosomes, cell debris, cell membranes, cell nuclei, cytoplasmiccomponents such as an endocytic compartment, golgi, endoplasmicreticulum and/or mitochondria. It further relates to stabilising RNA,DNA and/or proteins contained within exosomes derived from blood, serum,plasma, urine, cerebral spinal fluid and other bodily fluids forstorage, transport and handling for a diagnostic test such asextraction, analysis and/or identification of miRNA or mRNA containedwithin an exosome or other extracellular microvesicle.

In a further aspect the present invention provides use of a DES mixtureas a stabiliser of pre-purified particles and molecules such as viralparticles, bacteriophages, RNA, DNA, proteins such as enzymes andantibodies, lipids and fats and/or carbohydrates. Pre-purified isdefined here as a sample consisting of greater than 80% of the particleor molecule of interest.

In a further aspect, the present invention provides use of a DES mixtureas a stabiliser of biomolecules, bacteria, fungal and/or other pathogenssuch as Leishmania, Trypanosoma and/or Plasmodium in plasma, serum,buffy coat and/or blood for subsequent pathogen diagnostic analysisincluding bacterial and/or viral diagnostics.

In a further aspect, the present invention provides use of a DES mixtureas a stabiliser of viruses, viral particles, viral nucleic acids and/orproteins such as Influenza, HPV, HIV, HBV, HCV, Foot and Mouth DiseaseVirus, Influenza, SARS and/or West Nile Virus, in biological samplessuch as sputum, a swab, plasma, serum, buffy coat and/or blood forsubsequent viral diagnostic analysis.

In a further aspect, the present invention provides an apparatussuitable for cell or tissue fixation and/or for inhibiting thedegradation of a biomolecule, which apparatus comprises: a first deepeutectic solvent layer formed of a first deep eutectic solvent; and arecess in the first deep eutectic solvent layer for receiving a samplecomprising the biomolecule wherein the first deep eutectic solvent is asolid or a gel. The apparatus may further comprise a second deepeutectic solvent layer formed of a second deep eutectic solvent, whereinthe second deep eutectic solvent layer encloses the recess; and whereinthe second deep eutectic solvent is a solid or a gel.

The first and/or second deep eutectic solvent may be a deep eutecticsolvent as defined herein and preferably comprises a component selectedfrom 3,3,3-trifluoropropanamide, 2,2-difluoro-2-phenylacetamide, ureaand thiourea.

The first and/or second deep eutectic solvent may also comprise acomponent selected from choline chloride, butyrylcholine iodide, andN,N,N-trimethylglycine.

The apparatus suitable for cell or tissue fixation and/or for inhibitingthe degradation of a biomolecule according to the invention comprises afirst deep eutectic solvent layer formed of a first deep eutecticsolvent. The first deep eutectic solvent is a solid or a gel. Suitably,the components of the deep eutectic solvent are selected such that thedeep eutectic solvent is solid under the desired conditions of use, forexample, at a temperature of about 25° C. and a pressure of about 1 atm.Alternatively or additionally, the deep eutectic solvent may be blendedwith a matrix material which is a solid or a gel under the desired usageconditions.

Particularly preferred deep eutectic solvents for use as the first deepeutectic solvent include choline chloride:3,3,3-trifluoropropanamide,optionally in a molar ratio of about 1:2; cholinechloride:2,2-difluoro-2-phenylacetamide, optionally in a molar ratio ofabout 1:1; choline chloride:trehalose, optionally in a molar ratio ofabout 1:1 and butyrylcholine iodide:urea, optionally in a molar ratio ofabout 1:2.

If the apparatus comprises a second deep eutectic solvent, the first andsecond deep eutectic solvents may be the same or different. Preferably,the second deep eutectic solvent is selected from cholinechloride:3,3,3-trifluoropropanamide, optionally in a molar ratio ofabout 1:2; choline chloride:2,2-difluoro-2-phenylacetamide, optionallyin a molar ratio of about 1:1; choline chloride:trehalose, optionally ina molar ratio of about 1:1; and butyrylcholine iodide:urea, optionallyin a molar ratio of about 1:2.

In a further aspect, the present invention provides a kit wherein thedeep eutectic solvent is a deep eutectic solvent as defined herein.

The invention is not limited particularly to the stabilisation of anyone type of biomolecule, but the improvement of the quality andintegrity of RNA is often notable as it is one of the most fragilemolecules in the cell due to its length and chemical sensitivity toenvironmental aggression. Generally as a rule of thumb it can beconsidered that if the rRNA molecules of the cell are intact then themRNA, DNA, proteins, lipids, carbohydrates and small metabolites willalso be intact, however if the rRNA is degraded the other biomoleculesmay or may not also be degraded depending on the particular chemicalsensitivity of each biomolecule. There are some exceptions such as ifthe tissue or cell is particularly rich in certain enzymes such asRNases, DNases, lipases, esterases, proteases or phosphatases which willlead to specific degradation or modification of one particular class ofbiomolecules.

The RNA referred to can be found, derived or associated with a samplesuch as a virus, cell, circulating tumour cell, cerebrospinal fluid,bronchoalveolar lavage, serum, plasma, blood, exosomes, othermicrovesicles preserved samples such as FFPE blocks or sections,biopsies, solid or liquid tissues or other biological samples. It canalso be a nucleoside or nucleotide containing molecule such as a cAMP,ATP, GTP, monomers, dimers and oligomers of deoxy- and ribonucleotides,deoxy- or ribo-oligonucleotides, plasmid DNA, genomic DNA, mitochondrialDNA, RNA such as microRNA (miRNA), piRNA, siRNA, tRNA, viriods,circulating RNA, circular ss or ds non-coding RNA, hnRNA, mRNA, rRNAsuch as the 5S, 5.8S, 16S, 18S, 23S and 28S rRNA species, and viral RNAderived from for example HCV, West Nile Disease Virus, Foot and MouthDisease Virus, Influenza, SARS, or HIV RNA, and/or extracellular RNA(exRNA).

It can also be a molecule derived from a synthetic organic proceduresuch as an oligo-synthesizer, a mixture of RNA and DNA, a chimera of RNAand DNA, the product of an enzymatic reaction such as an in vitro RNAtranscription, amplified RNA (aRNA), ribozymes, aptamers, a PCRamplification, rolling circle amplification (RCA) or ligase chainreaction (LCR) an internal control standard or control RNA.

RNA analysis methods that would benefit from this invention include invitro or in vivo protein translation of mRNA templates, RNA dependentRNA polymerisation, DNA dependent RNA polymerisation, RNA spliceanalysis, RNA folding analysis, aptamer and ribozyme production, opticaldensity (OD) measurements, RNA:protein interaction studies, RNAelectrophoresis and sedimentation including molecular weight standards,RNA bioconjugates, RNA ligation, RNA folding studies, RNA footprinting,RNA NMR structural studies, RNA oligonucleotide synthesis, RNA in situ,in situ hybridization (ISH), fluorescence in situ hybridization (FISH),RNA sequencing, reverse transcription (RT), RT-PCR, RT-qPCR, nucleaseprotection assays, hybridisation techniques such as northern blotting,bDNA, and microarrays including the preparation of probes, fluorescentnucleic acid labelling, NASBA, RNAi, miRNA techniques such as extractionand quantification and those methods requiring quality control and/orquantitative or qualitative measurements of RNA.

Instability refers to an alteration in the molecular weight or analteration of the chemical structure of the RNA molecule, suchinstability is associated with handling, storage, transport and/or theactual analysis of the analyte molecule. Biomolecule instability isoften dependent on the activity of naturally occurring catabolic enzymesand in particular RNases which can substantially alter the molecularweight of the RNA or involve much smaller molecular weight alterationsof the original analyte molecule. Such RNases can have an origin eitherin the biological sample itself, for example they can be releasedprogressively following sample handling or released massively as aresult of poor handling of the tissue when for example it has beenfreeze thawed, a process that generally leads to the rupture ofintra-cellular vesicles containing proteases and nucleases thatconsequently flood into the cytoplasm leading to very high rates ofanalyte degradation. Alternatively, the degradative enzyme can come fromexternal contamination of the sample environment such as microbialcontamination or spoilage of the sample. Analyte instability isgenerally associated with a reduction in the sensitivity or performanceof the analytical procedure, whether the analyte is a protein, nucleicacid, carbohydrate, lipid or metabolite.

‘Degradation’ refers to the physical or chemical changes that occur as aconsequence of biomolecule and/or analyte instability. As some examplesof degradation related to nucleic acids, degradation can refer to thedeamination of nucleobases such as the conversion of cytosine to uracil,oxidation of nucleobases such as guanine, the loss of methyl groups frommethyl-cytosine, the loss of one or more nucleobases such as occursduring depurination, the cleavage of phosphodiester bonds leading tochain cleavage and the loss of one or more nucleotides from the bulk ofthe nucleic acid molecule. It does not refer only to changes of thesecondary or tertiary structure of the molecule.

‘Integrity’ refers to the intactness of a molecule and therefore is theopposite of degradation.

‘Substantial degradation’ refers to a sample that contains at least halfof the analyte molecules that have been cleaved or reduced in molecularweight by 5% or more. Methods to determine degradation are well knownand depend on the analyte molecule (see Sambrook et al., (1989)Molecular Cloning: A Laboratory Manual (2nd Ed.) Cold Spring HarborUniversity Press, NY). The determination of the molecular weight andtherefore the extent of degradation of nucleic acids is commonly carriedout using denaturing or native gel electrophoresis and may includeSouthern or Northern blotting with a labelled hybridisation probe. RNAquantification can include analysis using an RNA Chip and the AgilentBioanalyser 2100™ system and calculating the ‘RNA Integrity Number’(RIN). Nucleic acid degradation can also be conveniently quantified byQ-PCR for DNA, and RT-qPCR for RNA using for example a Lightcycler™(Roche) and suitable amplification probes. Calculating the RT-qPCRamplification ratios of 3′/5′ ends of an mRNA, frequently β-actin,following reverse transcription using an oligo dT primer is alsocommonly used to assess RNA degradation. Other methods include RNAseq ofthe entire mRNA content of a cell or tissue, or comparing the relativehybridisation signals of oligonucleotides representing 3′ to 5′ sites ofmRNA following analysis using Affymetrix® GeneChips®. Smaller single ordouble stranded nucleic acids of less than 100 nucleotides in lengthsuch as oligonucleotides and miRNA are most accurately quantified bymass spectrometry such as MALDI-TOF MS, this technique having the addedadvantage of being able to also determine degradation events that do notsignificantly alter the molecular weight of the analyte such asdepurination or deamination of nucleobases. Most miRNA analyses arecarried out by dedicated RT-qPCR. Despite the sophistication of themethods for determining the extent of RNA degradation, it is evidentthat certain mRNA sequences are far more sensitive to degradation thatothers and because the 18 and 28S rRNA are relatively stable todegradation, they are only a poor surrogate marker for the extent ofmRNA degradation. Accurately analysing mRNA degradation is currentlybest carried out using RT-qPCR as explained above. For a detaileddescription of methods to evaluate RNA degradation as a result of RNApurification methods see Muyal et al., (2009) Diag. Path. 4:9.

A ‘pure sample’ of RNA or DNA refers to a nucleic acid solution in waterwhere the OD260/280 ratio is 1.7 or above.

Although generally instability and degradation are associated with areduction in the overall molecular weight of the molecule under study(“the analyte”), it can, conversely, be related to an increase in themolecular weight of a complex that progressively aggregates during, forexample, storage. One example of the latter would be the complexation oraggregation of proteins onto nucleic acids during storage of a wholetissue or the chemical cross-linking of molecules during the processingof a sample such as with formalin fixed paraffin embedded tissue(“FFPE”).

‘Stabilisation’ refers to conditions that lead to an overall reductionin the amount of degradation of an analyte molecule compared with thecontrol. Such a control can be the conditions used without the use ofthe invention, for example storage without any stabilizer (FIG. 11), butthe rate of RNA degradation is generally too high to be a usefulcomparison, therefore a better control is the commercially availableRNAlater used according to the manufacturer's instructions (Cat. No.76106, Qiagen, Germany). The control may be an excised piece of tissuesuch as a biopsy, a blood or serum sample or a piece of tissue stored inRNAlater at for example 4, 20 or 37° C. for 1-16 Hrs, or 1-45 days.

Tissue disruption refers to the process of breaking a large tissuesample up into particles that are small enough to be consequently lysedby the addition of a chaotrope solution. The disruption breaks thesample up into pieces small enough to allow efficient release of theanalyte for consequent RNA extraction and purification.

An ‘RNase inactivation’ step can be carried out to sufficientlyinactivate RNase and allow subsequent stabilisation with a DES mixturewithout a significant degradation of the RNA. Numerous methods are knownin the art for inactivating RNases such as enzymatic degradation withproteases such as trypsin, chymotrypsin, papain or proteinase K,reduction of the disulphide bond with β-mercaptoethanol (Chirgwin, etal., “Isolation of Biologically Active Ribonucleic Acid from SourcesEnriched in Ribonuclease,” Biochemistry, 18:5294-5299, 1979),dithiothreitol, dithioerythritol, glutathione or TCEP, addition ofRNasin protein (Life Technologies, USA), RNAsecure™ (Life Technologies,USA), treatment with a chaotrope such as guanidine HCl, guanidinethiocyanate (Chomczynski and Sacchi, “Single Step Method of RNAIsolation by Acid Guanidine Isothiocyanate-Phenol-ChloroformExtraction,” Anal. Biochem., 162:156-159, 1987; Sambrook, et al.,“Molecular Cloning, A Laboratory Manual,” pp. 7.16-7.52, 1989), urea,formamide, formaldehyde or sodium iodoacetate, treatment with adetergent such as Tween-20, Triton X-100, NP-40, SLS, or SDS, EDTA,EGTA, sodium citrate, heat or acid denaturation, inhibition with vanadylribonucleoside complexes (Berger and Birkenmeier, 1979) or cross-linkingwith glutaraldehyde. The tissue sample is initially treated such thatRNase activity is reduced to at least 50, 75 or more preferably 100% ofits untreated activity prior to DES mixture treatment as set out in oneof the Examples here. Residual RNase activity prior to treatment can bemonitored using an RNaseAlert™ kit (Life Technologies, USA). Methods forinactivating RNases in tissues are set out in U.S. Pat. No. 6,777,210and US Patent Application Publication Number. 2009/0286304.

By way of example, but without limitation, the DES is made by mixing, ormixing and heating, one or more component(s) which may be chosen fromthe group: Choline nitrate, Choline tetrafluoroborate, Cholinehydroxide, Choline bitartrate, Choline dihydrogen citrate, Cholinep-toluenesulfonate, Choline bicarbonate, Choline chloride, Cholinebromide, Choline iodide, Choline fluoride, Chlorocholine chloride,Bromocholine bromide, lodocholine iodide, Acetylcholine hydroxide,Acetylcholine bitartrate, Acetylcholine dihydrogen citrate,Acetylcholine p-toluenesulfonate, Acetylcholine bicarbonate,Acetylcholine chloride, Acetylcholine bromide, Acetylcholine iodide,Acetylcholine fluoride, Chloroacetylcholine chloride, Bromoacetylcholinebromide, lodoacetylcholine iodide, Butyrylcholine hydroxide,Butyrylcholine bitartrate, Butyrylcholine dihydrogen citrate,Butyrylcholine p-toluenesulfonate, Butyrylcholine bicarbonate,Butyrylcholine chloride, Butyrylcholine bromide, Butyrylcholine iodide,Butyrylcholine fluoride, ChloroButyrylcholine chloride,BromoButyrylcholine bromide, lodoButyrylcholine iodide,Acetylthiocholine chloride, L-Carnitine, D-Carnitine, Betaine,Sarcosine, Trimethylamine N-oxide, Betaine HCl, Cetyl betaine,Cetyltrimethylammonium fluoride, Cetyltrimethylammonium chloride,Cetyltrimethylammonium bromide, Lauryl betaine,N,N-Dimethylenethanolammonium chloride, N,N-diethyl ethanol ammoniumchloride, Beta-methylcholine chloride, Phosphocholine chloride, Cholinecitrate, Benzoylcholine chloride, Lauryl sulphobetaine,Benzyltrimethylammonium chloride, Methyltriphenylphosphonium chloride,Methyltriphenylphosphonium bromide, Methyltriphenylphosphonium iodide,Methyltriphenylphosphonium fluoride, N,N-diethylenethanol ammoniumchloride, ethylammonium chloride, Tetramethylammonium chloride,Tetramethylammonium bromide, Tetramethylammonium iodide,Tetramethylammonium fluoride, Tetraethylammonium chloride,Tetraethylammonium bromide, Tetraethylammonium iodide,Tetraethylammonium fluoride, Tetrabutylammonium chloride,Tetrabutylammonium bromide, Tetrabutylammonium iodide,Tetrabutylammonium fluoride, (2-chloroethyl) trimethylammonium chloride,Terbium (III) chloride, Zinc (II) chloride, Zinc (II) bromide, Zirconium(III) chloride, Iron (III) chloride, Tin (II) chloride, Copper (II)chloride, Magnesium (II) chloride; with, one or more other component(s)that can also form a DES, including for example, but without limitation,one or more of the following chemicals chosen from the group: Urea,Formamide, Thiourea, 1-Methylurea, 1,1-Dimethylurea, 1,3-Dimethylurea,Carbohydrazide, Tetramethylurea, 1,3-bis(hydroxymethyl)urea, Benzamide,Girards Reagent T, Lactamide, Acetamide, Fluoroacetamide,Difluoroacetamide, Trifluoroacetamide, Chlorofluoroacetamide,Chlorodifluoroacetamide, Chloroacetamide, Dichloroacetamide,Dichlorofluoroacetamide, Trichloroacetamide, Bromoacetamide,Dibromoacetamide, Tribromoacetamide, Bromofluoroacetamide,Bromodifluoroacetamide, Bromochlorofluoroacetamide, Iodoacetamide,Diiodoacetamide, Triiodoacetamide, 2-Methyl-2,2-difluoroacetamide,2-Methyl-2-fluoroacetamide, 2,2-Dimethyl-2-fluoroacetamide,2-Ethyl-2,2-difluoroacetamide, 2-Ethyl-2-fluoroacetamide,2,2-Diethyl-2-fluoroacetamide, 2-Propyl-2,2-difluoroacetamide,2-Propyl-2-fluoroacetamide, 2,2-Propyl-2-fluoroacetamide,2-Fluoropropionamide, 3-Fluoropropionamide, 2,2-Difluoropropionamide,2,3-Difluoropropionamide, 3,3-Difluoropropionamide,3,3,3-Trifluoropropionamide, 2-Fluoro-3,3,3-trifluoropropionamide,2-Chloro-3,3,3-trifluoropropionamide,2,2-Chloro-3,3,3-trifluoropropionamide,2-bromo-3,3,3-trifluoropropionamide,2,2-Bromo-3,3,3-trifluoropropionamide, Pentafluoropropionamide,Heptafluorobutyramide, Trimethylacetamide, 1-(Trifluoroacetyl)imidazole,N,O-Bis(trifluoroacetyl)hydroxylamine, Bistrifluoroacetamide,N-Methyl-fluoroacetamide, N-Methyl-difluoroacetamide,N-Methyl-trifluoroacetamide, N-Methyl-chlorofluoroacetamide,N-Methyl-chlorodifluoroacetamide, N-Methyl-chloroacetamide,N-Methyl-dichloroacetamide, D N-Methyl-dichlorofluoroacetamide,N-Methyl-trichloroacetamide, N-Methyl-bromoacetamide,N-Methyl-dibromoacetamide, N-Methyl-tribromoacetamide,N-Methyl-bromofluoroacetamide, N-Methyl-bromodifluoroacetamide,N-Methyl-bromochlorofluoroacetamide, N-Methyl-iodoacetamide,N-Methyl-diiodoacetamide, N-Methyl-triiodoacetamide,N-Methyl-2-methyl-2,2-difluoroacetamide,N-Methyl-2-methyl-2-fluoroacetamide,N-Methyl-2,2-dimethyl-2-fluoroacetamide,N-Methyl-2-ethyl-2,2-difluoroacetamide,N-Methyl-2-ethyl-2-fluoroacetamide,N-Methyl-2,2-diethyl-2-fluoroacetamide,N-Methyl-2-propyl-2,2-difluoroacetamide,N-Methyl-2-propyl-2-fluoroacetamide,N-Methyl-2,2-propyl-2-fluoroacetamide, N-Methyl-2-fluoropropionamide,N-Methyl-3-fluoropropionamide, N-Methyl-2,2-difluoropropionamide,N-Methyl-2,3-difluoropropionamide, N-Methyl-3,3-difluoropropionamide,N-Methyl-3,3,3-trifluoropropionamide,N-Methyl-2-fluoro-3,3,3-trifluoropropionamide,N-Methyl-2-chloro-3,3,3-trifluoropropionamide,N-Methyl-2,2-chloro-3,3,3-trifluoropropionamide,N-Methyl-2-bromo-3,3,3-trifluoropropionamide,N-Methyl-2,2-bromo-3,3,3-trifluoropropionamide,N-Methyl-pentafluoropropionamide, N-Methyl-heptafluorobutyramide,N,N-Dimethyl-2,2,2-trifluoroacetamide, N-Ethyl-2,2,2-trifluoroacetamide,N,N-Diethyl-2,2,2-trifluoroacetamide,N-(Hydroxymethyl)Trifluoroacetamide, Ethyltrifluoroacetate,Dithiothreitol, Dithioerythritol, Beta-mercaptoethanol, Penicillamine,Tiopronin, Acrylamide, Methanol, Ethanol, Propanol, Butanol,Formaldehyde, Glutaraldehyde, Taurine, Aconitic acid, Adipic acid,Benzoic acid, Citric acid, Malonic acid, Malic acid, DL-Maleic acid,Oxalic acid, Phenylacetic acid, Phenylpropionic acid, Succinic acid,Levulinic acid, Tartaric acid, Gallic acid, p-Toluenesulphonic acid,Glycine, Alanine, Valine, Leucine, Isoleucine, Serine, Threonine,Tyrosine, Cysteine, Methionine, Aspartic acid, Asparagine, Glutamicacid, Glutamine, Arginine, Lysine, Histidine, Phenylalanine, Tryptophan,Proline, Ethylene glycol, Triethyleneglycol, Glycerol, Resorcinol,Phenol, 1,2-propanediol, 1,3-propanediol, 1,4-Butanediol,1,5-Pentanediol, 1,6-Hexanediol, 1,8-Octanediol, 1,12-Dodecanediol,m-Cresol, Imidazole, 1-Methylimidazole, 4-Methylimidazole,N-Methylpyrrolidone, N-Ethylpyrrolidone, N-Benzylpyrrolidone,2-imidazolindone, tetrahydro-2-pyrimidione, Guanidine, Guanidine HCl,Guanidine isothiocyanate, Guanidine sulphate, Ammonium acetate, Ammoniumbicarbonate, Ammonium chloride, Ammonium citrate dibasic, Ammoniumformate, Ammonium iodide, Ammonium nitrate, Ammonium phosphatemonobasic, Ammonium phosphate dibasic, Ammonium sulfamate, Ammoniumsulfate, Ammonium tartrate dibasic, Ammonium isothiocyanate, Ammoniumbenzoate, Ammonium bromide, Ammonium fluoride, Ammoniumhydrogensulphate, Ammonium trifluoroacetate, Ammonium thiosulphate,Adonitol, Ribitol, Rhamnose, Trehalose, D-Sorbitol, L-Sorbitol, Sorbose,Xylitol, Glucose, Sucrose, Lactose, Fructose, Maltose, Mannose,Mannitol, Arabinose, Galactose, Raffinose, Inositol, Erythritol orXylose.

It will be appreciated that some compounds disclosed herein may beionisable, i.e. some compounds may be weak acids, weak bases, orampholytes. Representations of the free forms of ionisable compounds areintended to encompass the corresponding ionised forms. For example,representations of carboxylic acid groups encompass the correspondingcarboxylate groups.

It should be noted that certain components such as Zinc chloride canform a DES with not only Choline Chloride but also Urea; both Cholinechloride and Zinc chloride appear in the same component group above,therefore certain DES mixtures can be prepared from chemicals within thesame group.

It should also be noted that the list above is non-exhaustive, and thatthe DES components are not particularly limited to any type of moleculeor property except hydrogen-bonding between DES components is frequentbut not an absolute requirement.

The deep eutectic solvents used in accordance with the present inventionare preferably substantially free of organic acids. In particular, thedeep eutectic solvents used in accordance with the present invention arepreferably substantially free of organic acids selected from malic acid,maleic acid, citric acid, lactic acid, pyruvic acid, fumaric acid,succinic acid, lactic acid, acetic acid, aconitic acid, tartaric acid,ascorbic acid, malonic acid, oxalic acid, glucuronic acid, neuramic acidand sialic acid.

Alternatively or additionally, R₆, R₇ and R₈ as shown in Formulae II andIII are selected such that the resulting compound does not comprise acarboxylic acid group. In a preferred arrangement, when R₆ is OH, R₇ isnot carbonyl. In another preferred arrangement, when R₇ is —Z—C(O)R₈, R₈is not OH.

The deep eutectic solvents used in accordance with the present inventionare preferably substantially free of metal salts. In one arrangement,the deep eutectic solvents are substantially free of metal salts otherthan salts of zinc or zirconium. The deep eutectic solvents arepreferably substantially free of NaH₂PO₄, Na₂HPO₄, NaHSO₃, Na₂SO₄,MgCl₂, CaCl₂, KCl, NaCl and Kl. If the deep eutectic solvent comprises asugar or sugar alcohol, the deep eutectic solvent particularlypreferably does not comprise a metal salt.

The deep eutectic solvents used in accordance with the present inventionare preferably substantially free of sugars or sugar alcohols selectedfrom sucrose, glucose, fructose, lactose, maltose, cellobiose,arabinose, ribose, ribulose, galactose, rhamnose, raffinose, xylose,sucrose, mannose, trehalose, mannitol, sorbitol, inositol, xylitol,ribitol, galactitol, erythritol, and adonitol. If the deep eutecticsolvent comprises a metal salt, the deep eutectic solvent particularlypreferably does not comprise a sugar or sugar alcohol.

It should also be noted that there is no particular maximum number ofDES components in the DES mixture, for example 2, 3, 4, 5, 6, 7, 8, 9,10 or more components can be mixed to produce a DES mixture, usually butnecessarily, in integer molar ratios for example in a two component DESmixture, component 1 and component 2 can be mixed in the followingratios: 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3,1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 (mol:mol) or for example in a threecomponent DES mixture, component 1, component 2 and component 3 can bemixed in these ratios: 10:1:1, 9:1:1, 8:1:2, 7:1:3, 6:1:4, 5:1:5, 4:1:6,3:1:7, 2:1:8, 1:1:9, 1:2:8, 1:3:7, 1:4:6, 1:5:5, 1:6:4, 1:7:3, 1:8:2,1:9:1, 1:10:1 (mol:mol:mol).

The skilled artisan will understand that other ratios of mixing thecomponents are also possible, for example 20:1 or 1:30 (mol:mol) andthat the particular molar ratio leading to the eutectic point is notnecessarily the optimum or desired molar ratio for the particularpurpose of, for example, stabilising RNA and/or fixing cell morphology.The stabilisation activity of the DES may be related to the hydrogenbonding properties of the DES mixture. There is no particularrestriction or limitation to the number of components or the ratio ofmixing to make a useful DES mixture for a particular application.Additional physical properties of the DES for example viscosity ordensity can be modified by either adding a further component, oralternatively, by adding an ‘additive’ as set-out below. Some DESmixtures can be prepared using a fractional molar ratio, for exampleZnCl2:urea has been prepared at 1:3.5 (mol:mol) ratio and there areagain no particular restrictions to the molar ratios of component 1 andcomponent 2, or component 1, component 2 and component 3 etc. that areused, and the optimum ratio of the components comprising the DES mixtureto be used for the application, such as cell fixation, is mostaccurately determined empirically. By way of example only, Cholinechloride/Trifluoroacetamide can be mixed in a 2:1, 1.75:1, 1.5:1,1.25:1, 1:1, 1:1.25, 1:1.5, 1:1.75 or a 1:2 mol:mol ratio, or furtherfractional amounts thereof, and subsequently individually tested todetermine which has the optimum RNA stabilization and/or cell fixationproperty.

Other factors such as the cost or toxicity may also lead the artisan toreduce the proportion of a particular component and to partially replaceit with a cheaper alternative. For example only, an Acetylcholinechloride:urea (1:2 mol:mol) DES mixture can, for certain applications bepartially replaced with the cheaper Choline chloride as in Acetylcholinechloride:Choline chloride:urea (1:1:4 mol:mol:mol).

It will also be apparent to the artisan that due to, for example, thepresence of contaminants in the components used to make the DES, such aswater, oxidation, absorption of CO2, contaminating by-products duringsynthesis or break-down products of one of the components, and that thedesired exact ratio, for example only, of a 1:1 (mol:mol) mixture maypractically mean+/−10%, but more preferably 5% and even more preferably1% difference in the exact amount of either component 1 or component 2.As way of illustration, a 5% error could potentially give the followingfinal molar ratios: 0.95:1, 1:0.95, 1.05:0.95 or 0.95:1.05 (mol:mol) andthe effect of this error can generally only be determined empirically,for example its effect on RNA quality as set-out in Example 1. Generallyin order to remove contaminants including water, the well-knownprocedure of recrystallisation in ethanol can be carried out followed byextensive vacuum and/or chemical drying. Methods are set-out in‘Purification of Laboratory Chemicals’ Butterworth-Heinemann (2012).

Typically, but certainly not exclusively, a DES is prepared by mixing ahydrogen bond acceptor from one of the following classes of chemicals;(i) a nitrogen salt with a positively charged cation such as primary,secondary, tertiary or quaternary nitrogen, one example of a nitrogensalt is one with a halide, such as Choline chloride, (ii) a metal saltsuch as a transition metal salt halide, with, a hydrogen bond donorwhich may include (iii) an amine, (iv) a hydroxyl, (v) an aldehyde, (vi)an amide, or (vii) carboxylic acid, hydrogen bond donors such as sugars,carboxylic acids, ureas such as Trifluoroacetamide, and alcohols wouldthus be included.

In one arrangement, the deep eutectic solvent is a type III or a type IVdeep eutectic solvent, wherein the RNA extracted from 10 mg of a ratliver sample incubated at 24° C. for 20 days with 400 mg of the deepeutectic solvent has an RNA integrity number (RIN) of at least 4.0 asmeasured using an Agilent Bioanalyser 2100.

RNA integrity number may be measured using an RNA 6000 Nano total RNAKit (Cat. No. 5067-1511, Agilent Technologies, USA) and a Bioanalyser2100 instrument (Cat. No. G2939AA, Agilent Technologies, USA)

As used herein, the term “type III deep eutectic solvent” refers to adeep eutectic solvent having at least a first component and a secondcomponent, wherein the first component is a quaternary ammonium orphosphonium compound such as choline chloride or N,N,N-trimethylglycine(betaine), and wherein the second component is a hydrogen bond donor,such as urea or trifluoroacetamide. A type IV deep eutectic solvent is adeep eutectic solvent comprising at least a first component and a secondcomponent, wherein the first component is a metal salt such as zincchloride and wherein the second component is a hydrogen bond donor, suchas urea.

A skilled artisan will be familiar with methods for extracting RNA fromtissue samples. Suitably, the RNA may be extracted using an RNeasy Minikit (Cat. No. 74106, Qiagen, Germany) in accordance with themanufacturer's instructions, although other methods may be used.

The present invention provides the use of a deep eutectic solvent as avirus, cell or tissue fixative to produce a fixed virus, cell or tissue.Preferably, at least 75% of HeLa cells grown on a substrate andincubated at 24° C. for 1 hour with the deep eutectic solvent remainattached following replacement of the deep eutectic solvent with waterand incubation at 24° C. for 1 hour.

A skilled artisan will be familiar with methods for determining thenumber of cells which remain attached to a substrate. For example,approximately 2,000 HeLa cells could be grown on a suitable substrate,such as a coverslip. One example of suitable coverslip is a Cellattice™:Micro-Ruled Cell Culture Surface with a diameter of 25 mm (Micro-ruledcell culture coverslip surface, Cat. No. CLSS-25D-050 NexcelomBioscience, USA). The substrate could be placed in a tissue cultureplate and grown overnight in an appropriate buffer, such as 2 ml ofDMEM/10% FBS. The number of attached cells in a defined area of thesubstrate may be counted manually using a 10× objective microscope lens.The tissue culture medium may be removed using, for example, anaspirating pipette and replaced with 400 mg of a deep eutectic solvent.The tissue culture may be incubated for 1 hour at room temperature toallow cell fixation and then the deep eutectic solvent removed replacedwith 2 ml of distilled water, incubated for 1 hour at room temperatureand the number of cells in a defined area of the grid counted manually.The percentage of attached cells remaining in the defined area comparedwith the original number may then be calculated.

In particularly preferred arrangements, the RNA extracted from 10 mg ofa rat liver sample incubated at 24° C. for 20 days with 400 mg of thedeep eutectic solvent has an RNA integrity number (RIN) of at least 4.0as measured using an Agilent Bioanalyser 2100 and at least 75% of HeLacells grown on a substrate and incubated at 24° C. for 1 hour with thedeep eutectic solvent remain attached to the substrate followingreplacement of the deep eutectic solvent with water and incubation at24° C. for 1 hour.

Optionally, the deep eutectic solvent used in accordance with thepresent invention is a type III deep eutectic solvent, wherein the deepeutectic solvent comprises a compound comprising a trifluoromethylgroup. The compound comprising the trifluoromethyl group may be presentin the deep eutectic solvent in an amount of at least 5% by weight ofthe deep eutectic solvent.

In one arrangement, the deep eutectic solvent comprises a firstcomponent and a second component, wherein first component is a compoundof Formula I:

-   -   wherein:        -   R₆ is H or OH;            -   R₇ is selected from H, CH₃, Cl, Br, a carbonyl oxygen,                and

-   -   Z is selected from —CH₂—, O and S;    -   R₈ is R₁₁ or OH; and    -   wherein the second component comprises a compound of Formula II        or a salt thereof:

-   -   wherein:        -   A is selected from 0, S, and NH;        -   R₁ is selected from H, an alkene group having 1 to 6 carbon            atoms, R₉, —NH₂, —NH—(CH₂)_(a)CH₃, and —C(R₃)(R₄)(R₅);            -   wherein a is 0 or an integer from 1 to 5;        -   R₂ is selected from H and linear alkyl group having 1 to 3            carbon atoms;        -   R₃ is an optionally substituted 5- or 6-membered aliphatic            or aromatic ring, wherein the substituent is R₁₀;        -   R₄ and R₅ are each independently H or F; and            wherein R₉, R₁₀, and R₁₁ are each independently selected            from alkyl groups having one to three carbon atoms,            monochloroalkyl groups having one to three carbon atoms, and            mono-, di- or tri-fluoroalkyl groups having one to three            carbon atoms.

Preferably, in this arrangement, A is selected from O, S, and NH; R1 isselected from H, —CH═CH2, R9, —NH2, —NHCH3, and —C(R3)(R4)(R5); R2 isselected from H and —CH3; R3 is an optionally substituted 5- or6-membered aliphatic or aromatic ring, wherein the substituent is R10;R4 and R5 are each independently H or F; and R9, R10, and R11 are eachindependently selected from alkyl groups having one to three carbonatoms, monochloroalkyl groups having one to three carbon atoms, andmono-, di- or tri-fluoroalkyl groups having one to three carbon atoms.Optionally, R7 is selected from H, Br, a carbonyl oxygen, and

A may be O or S. R₂ may be H.

R₁ may be selected from H, R₉, —CH═CH₂, and C(R₃)(R₄)(R₅). In thisarrangement, it is preferred that R₁ is R₉, wherein R₉ preferably hasone carbon atom.

Preferably, the second component is acetamide, or 2-chloroacetamide.

In another arrangement, R₉ is a mono-, di- or tri-fluoromethyl group.

The second component is preferably trifluoroacetamide,trifluorothioacetamide or N-methyltrifluoroacetamide.

In another arrangement, R₉ has two carbon atoms, preferably wherein R₉is a mono-, di- or tri-fluoroethyl group.

It is preferred that the second component is 2,2-difluoropropanamide or3,3,3-trifluoropropanamide. In another arrangement, the second componentis formamide or acrylamide.

In a further arrangement, R₁ is C(R₃)(R₄)(R₅), wherein R₄ and R₅ arepreferably F.

R₃ may be an optionally substituted 6-membered aromatic ring, such as anoptionally substituted phenyl group. Preferably, the first component is2,2-difluoro-2-phenylacetamide. Alternatively, R₃ comprises asubstituent, preferably at the 2-position of the phenyl group.

The substituent is preferably a mono-, di- or tri-fluoromethyl group.Thus, in one arrangement the second component is2-(trifluoromethyl)phenyl acetamide.

In a further arrangement R₁ is selected from —NH₂ and —NHCH₃.Preferably, the second component is urea, thiourea or 1,3-dimethylurea.

In a further arrangement, A is NH. In this arrangement the secondcomponent may be guanidine, optionally wherein the guanidine is presentin the form of a hydroisothiocyanate salt.

In a further arrangement, R₇ is H. In this arrangement the firstcomponent preferably comprises choline.

In another arrangement, the first component comprises bromocholine.

In another arrangement, the first component is N,N,N-trimethylglycine,optionally wherein the second component is selected fromtrifluoroacetamide and urea.

In another arrangement R₇ is

In this arrangement Z may be O or S and R₈ may be R₁₁, wherein R₁₁preferably has one carbon atom.

In this arrangement the first component preferably comprisesacetylcholine or acetylthiocholine.

Alternatively R₁₁ may have three carbon atoms, wherein the firstcomponent preferably comprises butyrylcholine.

In a further arrangement Z may be CH₂. In this arrangement, R₈ may beOH. In this arrangement, the second component is preferably carnitine.

The first component may comprise a counterion, which counterion istypically a halide anion but can be another anion such as nitrate (NO3-)or tetrafluoroborate (BF4-). The halide anion may be selected fromfluoride, chloride, bromide, and iodide, preferably chloride. In thisarrangement it is preferred that the first component is cholinechloride, optionally wherein the second component is selected fromtrifluoroacetamide, trifluorothioacetamide; 3,3,3-trifluoropropanamide;2,2-difluoro-2-phenylacetamide; thiourea and urea, preferablytrifluoroacetamide.

The molar ratio of the first component to the second component is in therange 1:3 to 2:1, preferably in the range 1:1.5 to 1:2.5, morepreferably 1:2.

In a further arrangement, the deep eutectic solvent comprises a firstcomponent and a second component, wherein the first component is ahalide salt of choline; and wherein the second component is anoptionally-substituted imidazole, wherein the or each substituent is analkyl group having 1 to 3 carbon atoms. In this arrangement, the molarratio of the first component to the second component is preferably inthe range 2.8:1 to 2:1.

The halide salt of choline is preferably choline chloride and thesubstituted imidazole is preferably a methyl imidazole, such asN-methylimidazole or 4-methylimidazole. The use of benzyl-substitutedimidazoles such as 1-benzylimidazole is also contemplated.Alternatively, the imidazole is unsubstituted.

In a further arrangement, the deep eutectic solvent comprises a firstcomponent and a second component, wherein the first component comprisesa compound of Formula III:

-   -   wherein:        -   R₆ is H or OH;        -   R₇ is selected from H, CH₃, Cl, Br, a carbonyl oxygen, and

-   -   -   Z is selected from —CH₂—, 0 and S;        -   wherein R₈ is selected from OH, an alkyl group having one to            three carbon atoms, a monochloroalkyl group having one to            three carbon atoms, and a mono-, di- or tri-fluoroalkyl            group having one to three carbon atoms; and

    -   wherein the second component is a sugar or a sugar alcohol        having at least 3 carbon atoms.

R7 is optionally selected from H, Br, a carbonyl oxygen, and

The sugar alcohol may be selected from glycerol, thrietol, xylitol,sorbitol and volemitol, preferably from glycerol, xylitol and sorbitol,and is most preferably sorbitol. Alternatively, the sugar may betrehalose.

In this arrangement, the second component may comprise choline, such ascholine chloride. The molar ratio of the first component to the secondcomponent may be in the range 1:2 to 2:1, preferably in the range 1:0.8to 1:1.2.

In a further arrangement, the deep eutectic solvent comprises a firstcomponent and a second component, wherein the first component is a zinc(II) halide or zirconium (IV) halide, and wherein the second componentis a compound of Formula IV:

-   -   wherein:        A is selected from 0, S, and NH;        R₁ is selected from H, an alkene group having 1 to 6 carbon        atoms, R₉, —NH₂, —NH—(CH₂)_(n)CH₃, and —C(R₃)(R₄)(R₅);        wherein n is 0 or an integer from 1 to 5;        R₂ is selected from H and linear alkyl group having 1 to 3        carbon atoms;        R₃ is an optionally substituted 5- or 6-membered aliphatic or        aromatic ring, wherein the substituent is R₁₀;        R₄ and R₅ are each independently H or F; and        wherein R₉ is selected from alkyl groups having one to three        carbon atoms, monochloroalkyl groups having one to three carbon        atoms, and mono-, di- or tri-fluoroalkyl groups having one to        three carbon atoms.

The preferred zinc (II) halide is ZnCl₂. The preferred zirconium (IV)halide is ZrCl₄. The second component is preferably urea. The molarratio of the first component to the second component may be in the range1:3 to 1:4.

The second component is preferably urea.

In a further arrangement, the deep eutectic solvent comprises a firstcomponent and a second component, wherein the first component is acompound of Formula V:

-   -   wherein:        -   Y⁻ is Cl⁻ or Br⁻;        -   X is N or P;        -   R₁₂, R₁₃, R₁₄ and R₁₅ are each independently a linear alkyl            group having 1 to 16 carbon atoms, a linear alcohol group            having 1 to 16 carbon atoms, a benzyl group, or a phenyl            group;    -   wherein the second component is a compound of Formula I or a        salt thereof:

-   -   wherein:        -   A is selected from 0, S, and NH;        -   R₁ is selected from H, —CH═CH₂, R₉, —NH₂, —NHCH₃, and            —C(R₃)(R₄)(R₅);        -   R₂ is selected from H and —CH₃;        -   R₃ is an optionally substituted 5- or 6-membered aliphatic            or aromatic ring, wherein the substituent is R₁₀; and        -   R₄ and R₅ are each independently H or F;    -   wherein R₉ and R₁₀ are each independently selected from alkyl        groups having one to three carbon atoms, monochloroalkyl groups        having one to three carbon atoms, and mono-, di- or        tri-fluoroalkyl groups having one to three carbon atoms.

Y⁻ may be Cl⁻. In alternative arrangements, Y⁻ may be any suitablecounterion, such as a halide, nitrate, or tetrafluoroborate.

X may be N; in this arrangement, the compound of Formula V is aquaternary ammonium salt.

Optionally, R₁₂, R₁₃, R₁₄ and R₁₅ are each independently a linear alkylgroup having 1 to 16 carbon atoms, a benzyl group, or a phenyl group

R₁₂, R₁₃, R₁₄, and R₁₅ may be each independently selected from linearalkyl groups having 1 to 4 carbon atoms, and are preferably each methylgroups. In this arrangement the first component is preferablytetramethylammonium chloride.

Alternatively, R₁₂, R₁₃, R₁₄, and R₁₅ are each ethyl groups. In thisarrangement the first component is preferably tetraethylammoniumchloride.

Alternatively R₁₂, R₁₃, R₁₄, and R₁₅ are each butyl groups. In thisarrangement the second component is tetrabutylammonium chloride ortetrabutylammonium bromide.

Alternatively, the first component comprises:

Alternatively, the first component comprises:

In another arrangement X may be P.

At least one of R₁₂, R₁₃, R₁₄, and R₁₅ may be a phenyl group. In thisarrangement the first component preferably comprisesmethyltriphenylphosphonium and may be methyltriphenylphosphoniumbromide.

In another arrangement A may be O or S.

R₁ may be selected from —NH₂ and —NHCH₃ and the second component ispreferably urea. Alternatively the second component may betrifluorothioacetamide. Alternatively the second component may betrifluoroacetamide.

The molar ratio of the first component to the second component may be1:1.5 to 1:2.5, preferably 1:1.8 to 1:2.2.

In a further arrangement the deep eutectic solvent comprises a firstcomponent and a second component, wherein the first component comprisescholine and wherein the second component is an alkanediol having 5 to 7carbon atoms. The alkanediol is preferably hexanediol.

In a still further arrangement the deep eutectic solvent comprises afirst component and a second component, wherein the first componentcomprises choline and wherein the second component comprises an N-alkylpyrrolidone, wherein the N-alkyl group has 1 to 5 carbon atoms.Preferably, the N-alkyl pyrrolidone is N-methylpyrrolidone. Preferably,the choline is choline chloride. In this arrangement, the molar ratio ofthe first component to the second component may be 1:2.

In another arrangement, the deep eutectic solvent comprises a firstcomponent and a second component, wherein the first component comprisescholine, and wherein the second component comprisesbeta-mercaptoethanol. In this arrangement, the choline may be cholinechloride and/or the molar ratio of the first component to the secondcomponent may be about 1:2.

In another arrangement, the deep eutectic solvent comprises a firstcomponent and a second component, wherein the first component comprisescholine, and wherein the second component comprises dithiothreitol. Thecholine may be choline chloride. The ratio of the first component to thesecond component may be about 1:2.

DES mixtures are most simply prepared from commercialised chemicalstocks available from chemical reagent companies such as Acros Organics(France), TCI (Belgium), Fluka (France) and the like. Typically thecorrect amounts of each component are added together in a polypropylenetube, briefly vortexed, heated to 100° C. by standard means and, ifnecessary for difficult to dissolve components or additives, sonicatedwhich can be an extremely effective method of mixing. Care should betaken with components such as Choline chloride which are hygroscopic,additional drying of the stock under vacuum or by recrystallisation maybe necessary. Stocks of DES mixtures can be kept dry under vacuum or byway of desiccants such as silica gel. Storage is generally at roomtemperature in a sealed tube.

It has also been found that treatment of plant and animal tissues with aDES mixture such as Choline chloride:Trifluoroacetamide (1:2 mol:mol)preserves the colour of leaves, flowers, blood and tissue, possibly byreducing oxidation compared with untreated samples or those treated withRNAlater, Formol or freezing. Colour preservation can be useful for thecorrect identification of specimens and/or components of specimens.

It has been found that the preparation of a DES mixture between Cholinechloride and volatile/odorous chemicals such as Dithiothreitol,Dithioerythritol, Beta-mercaptoethanol or Phenol notably reduces thevolatility and therefore the unpleasant or dangerous effects ofbreathing such chemicals. This effect is probably a result of hydrogenbonding between the volatile chemical and Choline chloride. There is noparticular limitation as to the combination of the volatile component inthe DES mixture, other than the necessity of hydrogen bonding betweenthe components. This is also a means to reduce the loss of othervolatile components such as low molecular weight alcohols, other organicchemicals such as solvents including DMSO and Formamide. The inventiontherefore also includes a means to reduce evaporation and/or volatilityof components for storage or use in an open environment. The advantagesinclude better health and safety, reduced loss of valuable reagents fromevaporation, reduced flammability and simpler disposal.

The eutectic point of a two or more component DES mixture is the pointwhere the proportion of component 1 relative to the other component(s)leads to the lowest melting temperature. For example the eutectic pointof a Choline chloride:Urea DES mixture is (1:2 mol:mol) with a reportedfreezing point (Fp) of 12° C. From a practical stand-point this meansthat the DES can be handled as a liquid at room-temperature but willslowly solidify in the fridge. Other DES mixtures, particularly thosecontaining Choline chloride mixed with Glycerol, Ethylene glycol andTrifluoroacetamide or Phenylacetic acid have much lower Fp, remainingliquid below 0° C. or even −20° C. Although handling liquids providescertain advantages such as ease of measuring exact volumes and mixing,it is important to note that this invention is not in any way limited toDES mixtures that are liquid at room-temperature of any other particulartemperature. Heating and mixing the components of a DES provides a meansfor individual atoms and molecules to establish hydrogen bonds or otherinteractions with other atoms and molecules and therefore produce theparticular property of the DES mixture. Once this has occurred, whetherthe DES mixture is a liquid, gel or solid does not necessarily changeits capacity to, for example, stabilise RNA in a tissue sample. Simplecontact between the tissue sample and the DES mixture will occur evenwhen the DES is a solid at room temperature and rapid stabilisation canoccur. Although traditionally RNA stabilisation reagents are liquidssuch as RNAlater, AllProtect, PAXgene Tissue or formalin, the inventiondescribed here can make use of the liquid, gel or solid DES RNAstabilisation and/or cell fixation properties. Indeed the use of a solidform of a DES can be advantageous when handling tissue samples; a solidblock of DES can be formed by heating the DES solid above its eutecticpoint so that it melts and then pouring it into a suitable receptacle tocool and solidify or, molded or cut to fit a specific container and thetissue sample placed on the solid DES surface to bring aboutstabilisation. Conveniently wells, depressions or grooves can be made inthe solid DES surface to serve as specific storage locations for eachsample, one well receiving one sample etc, advantageously with the useof a well, the surface area in contact between the solid DES and thesample is increased (FIG. 1, left), whilst an additional layer of a DESsolid or liquid above the sample located in the well will furtherincrease the total amount of DES available and surface area of contactwith the sample (FIG. 1, center). A blood collection tube is shown in(FIG. 1, right) containing a DES liquid, and with a pierceable cap forthe entry of the blood by syringe needle or blood collection kit(PreAnalytix). By way of example, DES mixtures that are solid or liquidat room-temperature are set out in Table 1.

The invention also includes the use of an additive in combination with aDES mixture. The purpose of the additive is to improve the properties ofthe DES mixture, for example, RNA, DNA and/or protein stabilisationand/or cell and tissue fixation or another application. The effects ofvarious additives on cell morphology for example are set out in Table 6.The purpose of the additive is to enhance the property of the DESmixture for the particular application, for example RNA stabilisation,storage, transport, and/or cell or tissue fixation. By way of exampleonly, the additive could be a (i) colourant or dye to aid in handling orstaining or processing the tissue such as H&E stain, Coomasie Blue,Methylene Blue, Xylene cyanol, Crystal violet, fuchsin, Acridine orange,DAPI, Carmine, Eosin, Ethidium bromide, Bismarck brown, Hoechst,Malachite green, Methyl green, Neutral blue, Nile blue, Osmiumtetroxide, Rhodamine or Safranin, (ii) detergent, quaternary ammoniumsalt or saponin to improve penetration of the cell plasma membrane withthe DES mixture, such as SDS, sodium lauryl sulphate (SLS),cetyltetrabutylammonium bromide (CTAB), tetrabutylammonium bromide(TBAB), sodium deoxycholate, Brij-35, Brij-58, NP-40, Triton X-100,Triton X-114, Tween-20, Tween-80, Octyl beta glucoside, CHAPS, Solanine,(iii) anti-microbial such as an antibiotic or antiseptic, such askanomycin, streptomycin or penicillin, sodium nitrate, sodium nitrite orsodium benzoate, (iv) protein precipitant such as Trichloroacetic acid,Ammonium sulphate, Sulphosalicyclic acid, Zinc salt such as ZnCl2 orZnSO4, (v) desiccant to remove excess water from the DES mixture such asMolecular sieves 4A, silica gel, CaCl2 or LiCl, (vi) a probe, ahybridising complementary nucleic acid, a peptide, protein, nucleic acidor labelled molecule, an internal control, a blocking sequence or acarrier nucleic acid such as (a) ss or ds RNA or DNA sequences, (b) apeptide, enzyme or other protein such as an antibody, (c) molecules fordetecting an analyte such as a biotin, horseradish peroxidase, avidin,streptavidin, fluorescent labelled molecule such as fluorescein, TexasRed, Alexa Fluor™ labelled LNA, (d) a Molecular Beacon™, a ScorpionProbe™, (vii) anti-oxidant to remove oxygen from the sample and reducedamaging oxidative effects during storage on for example the RNA or DNAnucleobases, such as Vitamin C or glutathionine, (viii) ribonucleaseinhibitor such as RNasin, SUPERase•IN™, RNaseOUT™, or a proteaseinhibitor such as phenylmethylsulfonyl fluoride (PMSF), diisopropylfluorophosphate (DFP), aprotinin or Pefabloc SC™, (ix) buffer tostabilise the pH between 5-8 or more preferably between 6 and 7, used inthe range 0.5-20 mM such as Tris-HCl, PIPES, MES, HEPES, MOPS, MOPSO,CAPS, CAPSO, BIPES, phosphate, imidazole, (x) chelator to removedivalent metal cations, used in the range 0.5-20 mM such as BAPTA, EDTA,EGTA, citric acid, D-Penicillamine, (xi) dissolved oxygen (in the finalconcentration range of 2-20%), and/or CO2 (in the final concentrationrange of 0.01-5%, a non-reactive gas such as Argon (in the finalconcentration range of 1-20%) and or a buffer, nutrients (such asglucose and amino-acids) to enhance cell, tissue and organism viability,and/or (xii) an alcohol in the range of 1-20% (wt:wt) to reduce DESviscosity such as a tert-butanol.

An ‘additive’ is defined here as any substance that can be dissolved ina particular DES mixture, and can be present in amounts greater than,equal to or less than the total amount of the DES mixture(weight:weight). By way of example only, Choline chloride:Urea:Zincchloride (1:2:0.5 mol:mol:mol), the ZnCl2 is an additive, or Cholinechloride: Trifluoracetamide:Urea (1:2:0.1 mol:mol:mol), the Urea is anadditive. It should also be noted that the additive does not necessarilyhave any particular effect on the freezing point of the DES mixture orform hydrogen bonds with any of the DES components but endows the DESmixture with a unique property.

A non-exhaustive list of possible additives to a DES mixture are:Ammonium p-toluenesulphonic acid, Sodium p-toluenesulphonic acid,Ammonium sulphate, Ammonium chloride, Ammonium thiosulphate, Sodiumdodecyl sulphate, Sodium lauryl sulphate, Sodium Benzoate,Dodecyldimethyl(3-sulphopropyl)ammonium hydroxide, Dimethylbenzenesulphonic acid, Congo Red, Giemsa, DAPI, Ethidium bromide, Mallory'sstain, Orcein, Aldehyde fuchin, Osmium tetroxide, Chromium trioxide,Chromic acid, Feulgen, Dichromate, Mercuric chloride, Haematoxylin andEosin stain (H&E), Formaldehyde, Glutaraldehyde, Acetone, Ethanol,Methanol, Methyltriphenylphosphonium bromide, Cetyltetrabutylammoniumbromide (CTAB), tetrabutylammonium bromide (TBAB), sodium deoxycholate,Brij-35, Brij-58, NP-40, Triton X-100, Triton X-114, Tween-20, Tween-80,Octyl beta glucoside, CHAPS, Solanine, kanomycin, streptomycin orpenicillin, sodium nitrate, sodium nitrite or sodium benzoate, ss or dsRNA or DNA sequences, ss or ds RNA or DNA labelled sequences, anaptamer, a peptide, enzyme, antibody, biotin, biotin labelled molecule,horseradish peroxidase, avidin, streptavidin, GFP or variant thereof,Luciferase, Fluorescein, Rhodamine, Texas Red, Alexa Fluor™ LNA,labelled LNA, a Molecular Beacon™, a Scorpion Probe™, FISH probe, bDNA,PCR primer, oligo (dT), PNA, anti-oxidant, RNasin, SUPERase•IN™,RNaseOUT™, phenylmethylsulfonyl fluoride (PMSF), diisopropylfluorophosphate (DFP), aprotinin or Pefabloc SC™, Tris-HCl, PIPES, MES,HEPES, MOPS, MOPSO, CAPS, CAPSO, BIPES, phosphate, imidazole BAPTA,EDTA, EGTA, citric acid, D-Penicillamine, O2, CO2, N2, Argon, propanol,tert-butanol, Trichloroacetic acid, sulphosalicyclic acid, Water,Methanol, Ethanol, Propanol, Butanol, Tetramethyl urea, Imidazole,1-Methylimidazole, 1-Ethylimidazole, 1-Benzylimidazole,4-Methylimidazole, N-Methylpyrrolidone, N-Ethylpyrrolidone,N-Benzylpyrrolidone, Guanidine, Guanidine HCl, Guanidine isothiocyanate,Ribitol, Rhamnose, Trehalose, D-Sorbitol, L-Sorbitol, Sorbose, Xylitol,Glucose, Sucrose, Lactose, Fructose, Maltose, Mannose, Mannitol,Arabinose, Galactose, Raffinose, Inositol, Erythritol, Xylose, Zincacetate, Zinc EDTA, Zinc phosphate, Zinc Trifluoroacetate, Zinc citrate,Zinc PISA, Zinc gluconate, Zinc chloride and/or Zinc sulphate.Non-dissolvable additives to DES mixtures include but not limited to:Paraffin, Silica gel, Sodium sulphate or Molecular Sieves™,Polyacrylamide, Aerogel, Polyacrylic acid and/or a Quantum Dot.

It has been found that the addition of ethanol or other alcohols such asmethanol and isopropanol to a Choline chloride:Trifluoroacetamide (1:2mol:mol) has a strong negative influence on the stability of RNA (seeTable 2). It is not understood why ethanol in particular has such anegative impact on RNA stability but a change in the Trifluoroacetamidehydrogen bonding with Choline chloride may be a partial explanation. Inany case it is preferable to avoid using ethanol at a molar ratiogreater than 0.1 compared with Component 1 (Choline chloride). However,if it is desired to process or store tissue samples that have beentreated with the DES mixture in ethanol or another alcohol, the excessDES mixture can be removed from the tissue sample using an absorbentpaper, washed two times in 100% ethanol before leaving the tissue forlonger periods, this abrogates the negative effect on RNA stabilisation.Alternatively the same method can be used to replace the first DESmixture with a second DES mixture, for example, a Cholinechloride:Trifluoroacetamide (1:2 mol:mol) mixture can be replaced with aCholine chloride:Urea (1:2 mol:mol) or another DES mixture which may bepreferable for certain applications involving RNA stabilisation.Alternatively the biological sample, once removed from the DES mixturecan be placed in a desiccating environment such as in a hermeticcontainer containing Molecular sieves, Drierite, a dry sugar (e.g.trehalose, xylitol or sorbitol), RNAstable® (Biomatrica, USA) or Calciumchloride for storage purposes. Yet another alternative is to place thetreated biological sample in a liquid storage medium such as alcohol(e.g. methanol, ethanol, propanol or butanol), RNALater (LifeTechnologies, USA), AllProtect (Qiagen, Germany), PAXgene tissue,PAXgene Blood, (PreAnalytix, Germany), GenTegra RNA (Integenx, USA),Cell-Free RNA BCT® (Streck, USA) or CellSave Preservative (Veridex,USA). As another alternative the sample, following treatment with theDES mixture can be transferred to a solution of a cross-linking fixativesuch as ten volumes of a 4% solution of formaldehyde or glutaldehyde andallowed to incubate for an appropriate time for example 15 minutes to 24hours. Although treating the sample with a cross-linking agent will havea negative effect on the quality of RNA, DNA and proteins, the treatmentmay improve the rigidity of the sample for slide preparation and itsperformance in certain assays such as immunohistochemistry employingantibodies specific for formalin treated protein epitopes. Anotheralternative is to pre-treat the sample with ten volumes of a 4% solutionof formaldehyde or glutaldehyde and allowed to incubate for anappropriate time for example 15 minutes to 24 hours, prior to removingthe sample and adding it to ten volumes of a DES mixture such as Cholinechloride:Trifluoroacetamide (1:2 mol:mol) for 15 minutes to 24 hours.

Preferably the use or presence of water is avoided in order to reducethe risk of analyte hydrolysis, in particular RNA, DNA and proteinhydrolysis. The DES mixture therefore contains 50% or less, morepreferably 40% or less, even more preferably 30% or less, even morepreferably 20% or less, even more preferably 10% or less, even morepreferably 5% or less and most preferably less than 2% water (weightpercentage) for applications requiring RNA, DNA or proteinstabilisation. For cell structure and morphology, and tissue fixationthe DES mixture may contain 50% or less, more preferably 40% or less,more preferably 30% or less, more preferably 20% or less, morepreferably 10% or less but most preferably between 10% and 5% water(weight percentage).

For optimum RNA stabilisation it has been found that the addition of adessicant to the Choline chloride:Trifluoroacetamide (1:2 mol:mol)mixture is preferable. Suitable desiccants include silica gel,polyacrylamide, Sodium sulphate, Drierite™ and Molecular sieves. Apreferred dessicant for RNA stabilisation is Molecular sieve Type 4A(powdered or in pellet form such as 4-12 Mesh) used at 5-50%weight:weight with the DES mixture, preferably a Cholinechloride:Trifluoroacetamide (1:2 mol:mol) mixture. An additionaladvantage is that it has been found that Molecular sieves reducecrystallisation occurring in the Choline chloride:Trifluoroacetamide(1:2 mol:mol) mixture.

It will be apparent that there are many types of DES mixture that can bemade and the choice of a particular DES for the purpose of preservingRNA in a sample will be determined by one or more of the followingpreferable features of the DES: (1) compatibility with RNA, preferablyit should have a pH between pH 4.5 and 8.5, more preferably between pH 5and 8, more preferably between pH 5.5 and 7.5 and most preferablybetween pH 6 and 7 when mixed with the sample, (2) compatibility withthe RNA purification reagents and protocol, for example it should notinterfere with the binding between the RNA and silica spin column suchas RNeasy (RNeasy Mini Kit, Cat. No. 74106, Qiagen, Germany) or alterthe partitioning of RNA in phenol containing reagents such as TRIzol(Cat. No. 15596018 Life Technologies, USA), (3) stabilising RNA in thetissue sample sufficiently quickly as to substantially alter theactivity of ribonucleases and/or accessibility of RNA to hydrolysis.Alternatively RNA is stabilised in the tissue sufficiently quickly thatthe RNA Integrity Number (RIN) is not reduced by more than 2 RIN units,more preferably not more than 1 RIN units, even more preferably not morethan 0.5 RIN units and most preferably less than 0.1 RIN units duringthe initial 18-24 hours storage with the DES mixture at room temperaturecompared with RNA extracted from a fresh tissue sample, without storage,of the same type, (4) have a capacity to stabilise RNA when the tissuerepresents at least 10%, more preferably 20% and even more preferably atleast 50% of the weight of the DES mixture (weight:weight), (5) notreduce RNA yield by more than 20% compared with fresh tissue, (6)provide stabilisation of DNA, proteins and the phosphate groups ofphospho-proteins, (7) be chemically stable, non-flammable, non-toxic tothe user and the environment, biodegradable, not react with bleach orreagents used during RNA purification to form toxic compounds, (8) havea shelf-life of at least 6 months at room temperature, (9) fix andstabilise native cell morphology and histology including antibodyepitopes and sub-cellular organisation and organelles, whilststabilising RNA and other biomolecules, (10) fix and stabilise cells insuch a way as to subsequently allow histological andimmunohistochemistry applications for example with antibodies and stainssuch as Hoechst or H&E stain, (11) stabilise RNA in FFPE samples toprotect and enhance the reversal of formalin cross linking attemperatures greater than 50° C. and to allow the melting and thereforethe easy removal of paraffin from the fixed sample, (12) fix andstabilise circulating tumour cells in whole blood to allow theirpurification, detection and molecular analysis.

The stabilisation of the RNA in the sample can be a result of DEStreatment, or a combination of DES treatment with another physicalprocess such as inactivation of ribonucleases, precipitation of cellularproteins and nucleic acid as a result of displacement of water moleculesor entry of the dissolved DES into the cell structure of the tissue andleading to RNA stabilisation or a combination of these. The modificationor alteration of hydrogen bonding in and around the cell and RNA, byDES's may be an important factor for the observed biomolecule and cellstabilisation as set out in this invention, however the exact mechanismby which DES mixtures can stabilise is not yet known.

The sample or tissue containing the analyte can be a (i) liquid such asblood, plasma, serum, cerebral spinal fluid (CSF), sputum, semen,bronchoalveolar lavage (BAL), amniotic fluid, milk and urine, (ii) solidsuch as body tissues (liver, spleen, brain, muscle, heart, oesophagus,testis, ovaries, thymus, kidneys, skin, intestine, pancreas, adrenalglands, lungs, bone and bone marrow), (iii) clinical for a medical testsuch as a prostate, breast or a cancer sample, tumour or biopsy,including a FFPE sample, circulating tumour cells, blood test, clinicalswabs, dried blood, exosome, microvesicles, (iv) animal tissues derivedfrom biomedical research or fundamental biology (monkey, rat, mouse,Zebra fish, Xenopus, Drosophila, nematode, yeast) and from their variousstages of development (egg, embryo, larvae, adult), (v) tissue andtissue culture cells used for drug discovery purposes, (vi) pathogenicand non-pathogenic microbes such as fungi, archaebacteria, gram-positiveand gram-negative bacteria, including E. coli, Staphylococcus,Streptococcus, Mycobacterium, Pseudomonas and bacteria that causeShigella, Diphtheria, Tetanus, Syphilis, Chlamydia, Legionella, Listeriaand leprosy, (vii) pathogenic or non-pathogenic viroids, bacteriophageor viruses that are found in a variety of biological samples such asbacteria, plants, blood, human tissues, animals blood, serum, plasma andtissues, and clinical samples, (viii) plants such as the leaves,flowers, pollen, seeds, stems and roots of rice, maize, sorghum, palm,vines, tomato, wheat, barley, tobacco, sugar cane and Arabidopsis, (ix)fixed tissue such as FFPE tissues and biopsies which frequently requirespecialised protocols for extracting high quality nucleic acids, (x)potentially pathogenic material associated with bioterrorism threatssuch as anthrax that may or may not need to be transported from thediscovery site to the testing facility, (xi) extremely small samplessuch as those derived from Laser Capture Microdissection samples (LCM),(xii) food samples that may for example contain food borne diseases,(xiii) soil sample. It should be noted that the sample may not bederived solely from biologically derived samples but also chemically orenzymatically synthesised ones such as nucleic acid based copiedmolecules or amplification products such as in vitro transcribed RNA andPCR products, oligodeoxyribonucleotides and oligoribonucleotides, PNAand LNA. There is no particular limitation to the type of sample thatcan be used with this invention.

The invention is also useful for the stabilisation of RNA, DNA andprotein internal controls (IC) and standards such as those included inHIV or HCV diagnostic kits such as Amplicor™ (Roche MolecularDiagnostics) or for carrier RNA that can be included in such diagnostickits. For this use, the RNA IC is commonly transported and stored withthe rest of the kit components, often at room temperature or 4° C. whichmay lead to degradation. Stabilisation of the RNA IC or carrier RNAimproves kit performance and maintains its integrity during transportand storage.

Usefully the invention can be used to preserve single and/or doublestranded RNA viruses including animal RNA viruses such as Norwalk,Rotavirus, Poliovirus, Ebola virus, Marburg virus, Lassa virus,Hantavirus, Rabies, Influenza, Yellow fever virus, Corona Virus, SARS,West Nile virus, Hepatitis A, C (HCV) and E virus, Dengue fever virus,toga (e.g. Rubella), Rhabdo (e.g. Rabies and VSV), Picorna (Polio andRhinovirus), Myxo (e.g. influenza), retro (e.g. HIV, HTLV), bunya,corona and reoviruses which have profound effects on human healthincluding viroid like viruses such as hepatitis D virus and plant RNAviruses and viroids such as Tobus-, Luteo-, Tobamo-, Potex-, Tobra-,Como-, Nepo-, Almo-, Cucumo-, Bromo-, Ilar-viruses, Coconutcadang-cadang viroid and potato spindle tuber viroid which all haveprofound effects on agricultural production are all liable to bedegraded before, during or after extraction for diagnostic detectionpurposes. The invention can also be used for stabilising single strandedRNA bacteriophage such as the genus Levivirus including theEnterobacteria phage MS2 and the genus Allolevivirus including theEnterobacteria phage Qβ, or double stranded RNA bacteriophage such asCystovirus including Pseudomonas phage φ6 or other types of phage suchas those used as internal RNA controls for diagnostic applications suchas those used in Armored RNA® (Ambion). The invention can also be usedfor stabilising internal control (IC) RNA or DNA sequences for use indiagnostic kits, by mixing a DES with a pure nucleic acid.

The invention can also be used to stabilise samples for the analysis ofmiRNA, siRNA and other small naturally occurring RNA molecules such assnRNAs, snoRNA, ncRNA, snoRNA, piRNA and rasiRNA. It can also be usedfor studies, diagnostics and therapies involving synthetic RNA of theRNAi type.

Usefully the invention can be used to preserve viral RNA such asretroviruses e.g. HIV, rotaviruses, HCV, and West Nile Virus in mixturesof guanidine and blood, serum, plasma cells and/or other medicallyimportant sample types such as cells and tissues.

It has been found that certain DES mixtures, notably Cholinechloride:Trifluoroacetamide (1:2 mol:mol) have a strong anti-microbialactivity (Example 34) thereby making the sample safer to work with andtransport. Other applications include stabilising and fixing soil and/orother environmental samples that may have been deliberately contaminatedwith toxins, viruses, bacteria and/or funghi for subsequent transportand laboratory analysis of RNA, DNA and/or proteins.

This invention therefore relates to methods to improve the storage,preservation, archiving and transport, to protect RNA from degradation,to increase their stability and as a consequence, to improve theanalytical sensitivity and assay quality.

It will be evident to one skilled in the art that various DES can betested for their suitability in this invention by adding them to thesample at a ratio of at least 10:1 (wt:wt) as set out in Example 1.Comparisons of the relative RNA stabilisation can be made using acontrol solution of RNAlater (10:1 (vol:wt) used according to themanufacturer's instructions (Cat. No. 76106, Qiagen, Germany). Followingincubation at, for example 37° C. for one day or more, the RNA isextracted according to a standard protocol such as RNeasy (RNeasy MiniKit, Cat. No. 74106, Qiagen, Germany) and its intactness analysed by gelelectrophoresis or by using a RNA 6000 Nano total RNA Kit (Cat. No.5067-1511, Agilent Technologies, USA) and Bioanalyser 2100 or othersuitable method such as RT-qPCR as described. RNA yields can bedetermined by OD260 nm uv spectrometry and purity by the well-knownOD260/230 and OD260/280 ratios. A Nanodrop ND2000 (ThermoScientificInc., USA) is one example of a suitable spectrometer for suchdetermination. Once suitable DES mixtures have been identified theiroptimum amount, purity, amount of contaminating water and use can bedetermined by further such tests.

Generally the most important single factor for optimisation is therelative molar ratios of the individual components in the DES mixture.In the first instance mixing only two components of the DES mixture isthe most straightforward means to identify approximately, and byempirical means as described above, components that result in thedesired effect, such as RNA stabilisation, yield, purity and suitabilityfor downstream applications such as RT-PCR or hybridisation. However itwill also be apparent to one skilled in the art that a blend of morethan two components may be desirable to further enhance or add novelproperties to the DES mixture. There are obviously a very large numberof potential mixtures of components that can lead to a DES mixture, andalthough it is easiest to start with the preparation of, in the firstinstance simple stoichiometric molar ratios of the DES components suchas, by way of example only, a 2:1, 1:1 or 1:2 molar ratio (mol:mol) ofcholine chloride and urea. It is thought that the depression in freezingpoint of two component DES mixtures is dictated, at least in part, bythe hydrogen bonding between the available hydrogen bond donor (e.g.choline chloride) and acceptor (e.g. urea). However it should be notedthat the preferred components of the DES mixture and their optimal ratioof mixing for a particular application such as RNA preservation or cellfixation is not necessarily related to the extent of the observedfreezing point depression. Identifying the optimum DES mixture for anapplication must therefore be determined empirically and consequentlytrial and error testing may be involved. It will be apparent thatidentifying the best blend for a particular purpose of a greater thantwo component DES mixture may involve significant effort. Therefore, inthe first instance and in the interest of rapidly defining suitablemolar ratios of components, stoichiometric ratios may be used asdescribed above. Once approximate molar ratios have been identified,further refinement involving fixing the molar amount of one componentwhilst varying the other two or more can be made. It should be notedthat the molarity of the various DES components is variable and dependson the density, molar ratios and molecular weight of the individualcomponents. By way of example, the Choline chloride concentration in aCholine chloride:Urea (1:2) mixture is approximately 5M and the Urea10M, compared with a Choline chloride concentration of 3.6M and aTrifluoroacetamide of 7.2M in a Choline chloride:Trifluoroacetamide(1:2) mixture.

The intactness of proteins and phospho-proteins can be determined bypreparative or analytical 2-D PAGE, mass spectrometry, suitableanti-phospho antibodies and ELISA as described in ProteomeCharacterization and Proteomics (2003) edited by Timothy D. Veenstra,Richard D. Smith.

Methods to recover the sample from a viscous DES liquid mixture includefixing or attaching it to a retriever such as a wire, a pin, a brush, abaton, a mesh, an insert, trapping it between two permeable membranes,or between two polymer pads containing sufficient DES to allow efficientDES stabilisation and/or fixation or using a shaped plastic holdercapable of gripping the sample during treatment. Preferably anycarryover of the DES mixture with the tissue sample is readily solublein the RNA lysis solution such as RLT (RNeasy Mini Kit, Cat. No. 74106,Qiagen, Germany), and either does not effect the RNA yield or increasesit, does not lead the sample to precipitate out of solution and if itcarries over with the RNA sample it has no effect on, or otherwiseenhances sensitive downstream applications such as RT-qPCR. Methods todetermine changes in RNA yield and quality are well known and includespectrophotometric methods, RNA 6000 Nano total RNA Kit (Cat. No.5067-1511, Agilent Technologies, USA) with Agilent Bioanalyser 2100quantification and/or RT-qPCR.

The results set out in this invention using DES mixtures areparticularly surprising as it is well known that nucleases have verybroad requirements for activity, for example Ribonuclease A is a robustnuclease which is active at pH 4-10, variable temperatures such as +4 to60° C., sodium concentrations of 0-3M, guanidine chaotropeconcentrations of up to 400 mM and can survive boiling for severalminutes (Raines (1998) Chem. Rev. 98:1045-1065). Despite being verydifficult to inactivate such enzymes, the treatment with many differentDES mixtures can have a profound effect on nuclease activity with aconsequent improvement in RNA quality. One explanation is that the DEScomponents hydrogen bond with enzymes such as nucleases and inparticular ribonucleases, displacing native water molecules around theenzyme and leading to denaturation and inactivation, and/or the RNA isprotected within denatured DES treated ribonucleoprotein complexes suchas ribosomes.

It will be apparent to one skilled in the art that testing theefficiency of a particular DES for biomolecule storage can be carriedout in a number of ways. Firstly a known amount of DES can be added to aset amount of pre-weighed tissue for example 20 mg frozen-thawed ratliver and incubated together for varying amounts of time for example 5,10, 20, 40 and 60 hours at 37° C. and after incubation the tissuerecovered and the RNA or other biomolecule recovered by purification andanalysed for intactness. The RNA quality can be determined for example,by RT-qPCR or Bioanalyser 2100 (Agilent, USA) RIN analysis.

Preferably the DES mixture has a reasonable shelf-life followingpreparation meaning that its stabilisation activity does notsignificantly change during storage for at least six months or more atambient temperature.

Agitation, mixing and temperature of the DES mixture with the tissuesample are all important factors for optimal RNA stabilisation. Gentleagitation of the DES mixture around the tissue sample can help toincrease the rate of stabilisation of the sample. Means of mixing oragitating include using the mixing function of the Thermomixer(Eppendorf, Germany), a rotating wheel or platform such as a LabRoller(LabNet, USA). Although the amount of RNA degradation observed using aDES mixture is limited, inevitably some degradation occurs during thetime delay between when the sample is removed from its normalenvironment in the whole animal and when the DES mixture can start toreduce or completely inhibit the ribonuclease activity. With respect tothis invention a key step is the time delay between adding the DESmixture to the sample and ribonuclease inactivation, and it will beevident that ribonucleases are more active at temperatures of 25-37° C.than at 4-16° C. Precooling of the DES and container and maintainingthis reduced temperature during the critical DES treatment step ispreferred although not essential for tissues with average levels ofribonuclease such as rat liver compared with tissues containing higherlevels such as the rat pancreas. RNA quality may be improved in tissuesthat have high ribonuclease activity by using a reduced temperature,such as 4° C., of the DES mixture, container and environment.

The appropriate choice of a DES mixture for preserving RNA in biologicalsamples depends on multiple overlapping physical and chemicalproperties. Ideally a DES mixture should be capable of: (1) rapidly andefficiently stabilising biomolecules including RNA, DNA, proteins,post-translationally modified proteins such as phospho-proteins,carbohydrates, lipids and metabolites in a biological sample, (2)functioning optimally to stabilise biomolecules such as RNA, when addedin a 20:1 (weight:weight) ratio, more preferably 15:1, even morepreferably 10:1, even more preferably 8:1, even more preferably 6:1 evenmore preferably 4:1, even more preferably 3:1, even more preferably 2:1and most preferably a 1:1 ratio with the biological sample, (3) being aliquid at below 120° C., even more preferably below 100° C., even morepreferably below 80° C., even more preferably below 60° C., and mostpreferably at room temperature, (4) being not so viscous that workingwith it and/or removing it from a solid sample is particularly difficultand have a viscosity at 25° C. of less than 100000 cP, even morepreferably less than 75000 cP, even more preferably less than 50000 cP,even more preferably less than 25000 cP, even more preferably less than10000 cP, even more preferably less than 5000 cP, even more preferablyless than 2500 cP, even more preferably less than 1 000 cP, even morepreferably less than 750 cP, even more preferably less than 500 cP, evenmore preferably less than 250 cP, even more preferably less than 100 cP,even more preferably less than 75 cP, even more preferably less than 50cP, even more preferably less than 25 cP, even more preferably less than10 cP and most preferably less than 5 cP (5) compatibility with RNA andRNA purification reagents such as guanidine HCl, guanidine thiocyanate,Lysis buffer RLT (RNeasy Mini Kit, Cat. No. 74106, Qiagen, Germany)silica spin columns, magnetic silica beads such as MagNA Pure® (RocheApplied Science, UK), TRIzol® (Life Technologies, USA), (6) not reducingRNA binding to a silica column such as RNeasy (Qiagen, Germany) by morethan 20% and not decrease the OD260/280 nm spectrophotometric ratio bymore than 0.2 compared with a standard purification, and (7) beingnon-toxic and non-flammable to the user even in combination with the RNApurification reagents, (8) biodegradation, and (9) non-volatility.

As one example of the use of the DES for RNA extraction from FFPEsamples, it is well known that formalin fixation leads to multiplecross-linking between the RNA and proteins making subsequent RNAextraction problematic and the quality of the RNA low. The standardprotocol for removing cross-links from RNA in a FFPE sample is to heatit, for example at 80° C. for 15-60 minutes, however this causes furtherRNA degradation (RNeasy FFPE Kit, Cat. No. 73504, Qiagen, Germany). Bycombining DES with the reagents necessary for the reversal ofcross-links, the RNA can be protected during the essential heating stepleading to a better quality RNA sample. As one example of a suitableDES, Choline chloride:Trifluoroacetamide (1:2) may be used to treat theFFPE sample at 80° C. to remove paraffin and cross-links from the RNA.

As another example of the use of DES in the medical field is for thetreatment of whole blood in order to stabilise circulating tumour cells(CTC's). These cells that are derived from the tumour and then enter theblood stream and are increasingly being used for diagnostic, prognosticand therapeutic purposes and also as end-points for numerous clinicaltrials of chemotherapeutic drugs. However, stabilising these cells inwhole blood is problematic so that shipping of patient blood may lead toloss or difficulty in correctly identifying CTC's and/or carrying outmolecular diagnostic analyses. Using a DES mixture to stabilise theCTC's in whole blood in vitro overcomes many of these drawbacks to thecurrent technology of CTC preservation. By way of example only, CTC's inwhole blood may be treated with at least six volumes of Cholinechloride:Trifluoroacetamide (1:2 mol:mol) in order to fix the cells andpreserve the RNA. The CTC's can then be subsequently collected andpurified using any one of a number of different methods such asCellSieve™ (MD, USA).

The invention will be described in further detail, by way of exampleonly, with reference to the following Examples and Drawings.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 shows apparatus according to the invention in which a DES mixtureis disposed in capped vials;

FIG. 2 shows the results of agarose gel electrophoresis on RNA samplesextracted according to the invention and the prior art;

FIG. 3 shows the results of agarose gel electrophoresis on RNAstabilised in a DES according to the invention at a range oftemperatures;

FIG. 4 shows the results of agarose gel electrophoresis on RNA preservedin tissue according to the invention or the prior art;

FIGS. 5A and 5B show the results of agarose electrophoresis on RNAstabilised in mouse tissue samples using a DES according to theinvention and the prior art;

FIG. 6 shows the results of agarose gel electrophoresis on RNA preservedin varying amounts of tissue according to the invention and the priorart;

FIG. 7 shows the results of agarose gel electrophoresis on RNA purifiedfrom DES-stabilised whole blood spiked with HeLa cells;

FIG. 8 shows the results of agarose gel electrophoresis on RNAstabilised in whole blood spiked with HeLa cells for 18 hours prior toextraction;

FIG. 9 shows the stabilization of RNA in whole blood with eitherguanidine or choline chloride:trifluroacetamide;

FIG. 10 shows light microscope images of HeLa cells fixed with cholinechloride: trifluoroacetamide according to the invention;

FIG. 11 shows the results of agarose gel electrophoresis on RNA degradedin tissue samples in the absence of any stabiliser;

FIG. 12 shows the results of agarose gel electrophoresis on genomic DNAstabilised in HeLa cells according to the invention or the prior art;

FIG. 13 shows the results of SDS-polyacrylamide gel electrophoresis onprotein from mouse liver stabilised according to the invention or priorart;

FIG. 14 shows the results of agarose gel electrophoresis on DNA and RNAfrom stabilised HeLa cells and optionally subjected to a processing stepbefore purification;

FIG. 15 shows the results of agarose gel electrophoresis on RNA and DNAwhose integrity are measured after fixation according to the inventionor prior art;

FIG. 16 shows the results of agarose gel electrophoresis on RNA fromDrosophila melanogaster embryos treated in accordance with the inventionor the prior art; and

FIG. 17 shows the results of agarose gel electrophoresis on RNA fromAllium cepa leaf shoots stabilised according to the invention or theprior art.

EXAMPLES 1. RNA Stabilisation in Animal Tissue Samples

To 400 μl of Choline chloride:Urea (1:2 mol:mol) in a standard 1.5 mlpolypropylene microcentrifuge tube was added 2-25 mg rat liver sampleand pre-incubated for 20 minutes at room temperature to allowstabilisation and/or fixation, the sample can then be incubated in theDES mixture at −80, −20, 4, 20 or 37, 42 or 55° C. for one hour toseveral weeks prior to recovery of the tissue sample with forcepsfollowed by RNA purification as set out below.

The rate of fixation for some dense, air filled and/or problematictissues can be improved using a vacuum system such as a Nalgene handheld vacuum pump (Cat. No. 6132-0020, ThermoScientific, UK) duringfixation.

The sample is then added to a fresh tube containing 350 μl of Lysisbuffer RLT, the tissue homogenised according to manufacturer'sinstructions (RNeasy Mini Kit, Cat. No. 74106, Qiagen, Germany). 300 μlportions of the lysate were then purified immediately according tomanufacturer's instructions and eluted into 20-50 μl of water. The yieldand purity of the RNA was then compared by OD 260/280 nm and theintegrity of the RNA determined by RT-qPCR using oligo dT cDNA primingand β-actin PCR primers (QuantiTect SYBR Green PCR Kit, Cat. No. 204141,Qiagen, Germany) and a LightCycler (Roche Applied Science, France) or byobtaining the RNA Integrity Number (RIN) by using an RNA 6000 Nano totalRNA Kit (Cat. No. 5067-1511, Agilent Technologies, USA) and aBioanalyser 2100 instrument (Cat. No. G2939AA, Agilent Technologies,USA).

Other types of commercialised RNA purification kits can replace theRNeasy kit and there is no particular limitation to the type of kitused.

The liver sample can be replaced with other tissue and cell types suchas liver, spleen, brain, muscle, heart, oesophagus, testis, ovaries,thymus, kidneys, skin, intestine, pancreas, adrenal glands, lungs, bonemarrow or cells such as COS-7, NIH/3T3, HeLa, 293, and CHO cells or evenliquid samples such as serum, plasma or blood.

FIG. 2. A 1% agarose-EtBr gel electrophoresis image of 300 ng RNAsamples extracted using an RNeasy kit: Lanes 1-5; Choline chloride:Urea(1:2 mol:mol) stabilised or lanes 6-10 RNAlater (Cat. No. 76106, Qiagen,Germany) stabilised 15 mg rat liver stored for 5 minutes (lanes 1+6), 1day (lanes 2+7), 3 days (lanes 3+8), 7 days (lanes 4+9) or 21 days(lanes 5+10). It can be observed that after storage at 37° C. that thereis improved RNA stability with Choline chloride:Urea stabilised samplescompared with RNAlater, by way of comparison after 7 days, the RNAIntegrity Number for Choline chloride:Urea sample was RIN=8, whilst thatfor RNAlater stabilised samples the RIN=5.10.

2. RNA Stabilisation in Animal Tissue Samples Using Other CholineChloride Based DES Mixtures

To 400 μl of Choline chloride:Trifluoroacetamide (1:2 mol:mol) orCholine chloride:Sorbitol (1:1 mol:mol) in a standard 1.5 mlpolypropylene microcentrifuge tube was added 2-25 mg rat liver sampleand pre-incubated for 20 minutes at room temperature to allowstabilisation and/or fixation, the sample can then be incubated at −80,−20, 4, 20 or 37, 42 or 55° C. for one hour to several weeks prior torecovery of the tissue sample with forceps followed by RNA purificationas set out in the following example.

The sample is then added to a fresh tube containing 350 μl of Lysisbuffer RLT, the tissue homogenised according to manufacturer'sinstructions (RNeasy Mini Kit, Cat. No. 74106, Qiagen, Germany). 300 μlportions of the lysate were then purified immediately according tomanufacturer's instructions and eluted into 20-50 μl of water. RNA yieldand quality was determined as set out in Example 1 and Table 1 and forboth DES mixtures RNA integrity was superior compared with RNAlater.

An appropriate source of Choline chloride is Cat. No. 110295000, AcrosOrganics, France, Sorbitol is Cat. No. S0065, TCI, Belgium andTrifluoroacetamide is Cat. No. T0598, TCI, Belgium.

3. RNA Stabilisation in Animal Tissue Samples Using Other DES Mixtures

To 400 μl of the following DES mixtures in a 2 ml polypropylene tube wasadded 5-15 mg of rat tissue (frozen tissue stock), following a 20 minutefixation step the sample was incubated at 37° C. for 18 hours prior toRNA extraction/purification using a RNeasy Micro kit (Cat. No. 74004,Qiagen, Germany). Whether the DES mixture was a solid or liquid at roomtemperature, the RNA yield following extraction and the RNA quality on ascale of 1-10 (with 0 indicating no stabilisation and 10 indicating nodegradation, compared with the RNA quality of immediately extracted RNAfrom a fresh rat liver tissue sample=10). Results are shown in Table 1below (nd=not determined, ‘Saturated’ solution is not a DES).

TABLE 1 RNA stabilisation in animal tissue using a two-component DESMixtures. RNA Ratio Liquid at RNA Yield Quality Component 1 Component 2mol:mol 24° C. ng/μl 0-10 1 Choline chloride Urea 2:1 No 120 8 2 Cholinechloride Urea 1:1 No 185 7 3 Choline chloride Urea 1:2 Yes 221 7 4Choline chloride Water 6M Saturated 42 3 5 Water Urea 5M Saturated 0 0 6Choline chloride Glycerol 1:2 Yes 62 4 7 Choline chloride Ethyleneglycol 1:2 Yes 26 3 8 Choline chloride Hexanediol 1:2 No 94 8 9Acetylcholine chloride Urea 1:2 Yes 156 9 10 Acetylcholine chlorideTrifluoroacetamide 1:2 Yes 76 5 11 Choline chloride Malonic acid 1:1 Yes152 1 12 (2-Chloroethyl) trimethylammonium Urea 1:2 No 172 5 chloride 13Choline chloride Trehalose 1:1 No 285 7 14 Choline chloride Xylitol 1:1Yes 236 9 15 Choline chloride Sorbitol 1:1 Yes 395 9 16 Choline chlorideGuanidine isothiocyanate 1:2 No 75 7 17 Urea Guanidine isothiocyanate1:2 No 10 0 18 Choline chloride Phenylacetic acid 1:1 Yes 250 3 19Choline chloride ZnCl2 1:2 No 173 7 20 Carnitine Trifluoroacetamide 1:2Gel 87 5 21 Taurine Trifluoroacetamide 1:2 No 6 4 22 Tetramethylammonium chloride Urea 1:2 No 80 7 23 Tetraethyl ammonium chloride Urea1:2 No 70 7 24 Tetrabutyl ammonium bromide Urea 1:2 No 86 5 25Tetrabutyl ammonium iodide Urea 1:2 No 12 1 26 Tetramethyl ammoniumoxide Trifluoroacetamide 1:2 Yes 19 2 27 Choline chloride Imidazole 7:3No 82 6 28 Cetyltrimethylammonium bromide Urea 1:2 No 65 4 29Cetyltrimethylammonium chloride Trifluoroacetamide 1:2 Yes 15 6 30 CaCl2Urea 1:3.5 No 116 2 31 ZrCl4 Urea 1:3.5 No 154 8 32 TbCl3 Urea 1:3.5 No0 0 33 ZnCl2 Urea 1:3.5 Yes 162 5 34 ZnCl2 Trifluoroacetamide 1:3.5 No15 6 35 Choline chloride N-methylpyrrolidone 1:2 Yes 86 6 36 Cholinechloride Acetamide 1:2 No 139 6 37 Choline chloride Thiourea 1:2 No 2297 38 Butyrylcholine iodide Urea 1:2 No 240 7 39 Acetylthiocholinechloride Urea 1:2 No 165 6 40 Choline bromide Urea 1:2 No 122 7 41Choline bromide Trifluoroacetamide 1:2 Yes 129 7 42 Choline chlorideAcrylamide monomer 1:2 Gel 164 7 43 Choline chloride 2-Chloroacetamide1:2 Yes 196 5 44 Choline chloride Bistrifluoroacetamide 1:2 Yes 2 0 45Choline chloride 2,2-Difluoropropanamide 1:2 Yes 191 6 46 Cholinechloride 2,2,2-Trifluorothioacetamide 1:2 Yes 1183 7 47 Choline chloride2-(Trifluoromethyl) 1:2 No 94 4 phenylacetamide 48 Choline chloride2,2-Difluoro-2-phenylacetamide 1:1 No 338 7 49 Choline chloride2,2,2-Trifluoro-N- 1:2 No 91 1 phenylacetamide 50 Choline chloride3,3,3-Trifluoropropanamide 1:2 No 404 8 51 Choline chloride Formamide1:2 Yes 81 5 52 Choline chloride Beta-Mercaptoethanol 1:2 Yes 395 7 53Choline chloride Dithiothreitol 1:2 Yes 202 7 54 Choline chlorideDithioerythreitol 1:2 Yes 109 2 55 Choline chloride Tiopronin 1:2 Yes287 1 56 Choline iodide Urea 1:2 No 89 4 57 Choline dihydrogen citrateUrea 1:2 Yes 67 3 58 Choline bitartrate Urea 1:2 No 58 4 59 Bromocholinebromide Urea 1:2 No 134 6 60 Choline chloride 1,3-dimethylurea 1:2 No127 6 61 Choline chloride Carbohydrazide 1:2 No 129 2 62 Cholinechloride 1,3-bis(hydroxymethyl)urea 1:2 No 0 0 63 Choline chlorideN-Methyltrifluoroacetamide 1:2 No 40 7 64 Choline chlorideDimethyltrifluoroacetamide 1:2 Yes 22 4 65 Choline chlorideDiethyltrifluoroacetamide 1:2 Yes 67 3 66 Choline chloride (1-trifluoro)acetylimidazole 1:2 Yes 22 1 67 Choline chloride Ethyl trifluoroacetate1:2 Yes 43 0 68 Choline chloride Pentafluoropropionamide 1:2 No 62 4 69Choline chloride Heptafluorobutyramide 1:2 No 10 4 70 Choline chlorideN-Methylbis(Trifluoroacetamide) 1:2 No 14 1 71 Choline chlorideLactamide 1:2 Yes 20 7 72 Choline chloride 2-Bromoacetamide 1:2 No 23 473 Beta-methylcholine chloride Trifluoroacetamide 1:2 Yes 19 7 74Betaine Urea 2:1 No 171 3 75 Betaine Urea 1:1 Yes 175 6 76 Betaine Urea1:1.75 Yes 208 6 77 Betaine Urea 1:1.95 Yes 30 7 78 Betaine Urea 1:2 Yes229 7 79 Betaine Urea 1:2.14 Yes 169 7 80 Betaine Urea 1:2.34 Yes 100 681 Betaine Urea 1:3 No 86 4 82 Betaine Urea 1:4 No 63 3 83 Betaine ZnCl22:1 No 52 4 84 Betaine Water Sat'd Yes 0 0 85 Betaine Trifluoroacetamide1:2 Yes 256 8 86 Carnitine Urea 1:2 Yes 217 5 87 Girards reagent T Urea1:2 Yes 72 1 88 Benzyltrimethylammonium chloride Urea 1:2 No 136 8 89Benzyltrimethylammonium chloride Trifluoroacetamide 1:2 Yes 35 6 90Methyltriphenylphosphonium bromide Ethylene glycol 1:3 Yes 106 5 91Methyltriphenylphosphonium bromide Trifluoroacetamide 1:2 Yes 207 4 92Choline chloride Trifluoroacetamide 1:2 Yes 74 9 93 Choline chlorideTrichloroacetamide 1:2 No 29 3 94 Urea Guanidine isothiocyanate 1:2 No10 0 95 Formaldehyde (4%) — — — 4 0

Qualitative RNA quality scale as follows; 0 (highly degraded) to 10(highest quality). The RNA analysis in Table 1 and 2 was carried out asfollows; ethidium bromide stained, 1% agarose 0.5×TAE gelelectrophoresis followed by visual analysis of a photograph taken underuv light, of the integrity of the 18S and 28S rRNA bands. An RNA samplewith an RNA Quality score of 8 or more has an 18S to 28S rRNA ethidiumbromide staining ratio of 1:2, whilst an RNA sample with an RNA Qualityscore of 5 has an 18S to 28S rRNA staining ratio of approximately 1:1.

TABLE 2 Two-Component DES Mixtures plus additive Additive Ratio (moleratio) relative to RNA Yield RNA Component 1 Component 2 (mol:mol)Component 1) (ng/μl) Quality 1 Choline chloride Urea 1:2 — 168 8 2Choline chloride Urea 1:2 Ammonium p-toluenesulphonic 246 6 acid (0.8) 3Choline chloride Urea 1:2 Sodium p-toluenesulphonic acid 60 7 (0.8) 4Choline chloride Urea 1:2 Dimethylbenzene sulphonic acid 126 7 (0.8) 5Choline chloride Urea 1:2 (NH4)2SO4 (0.015) 295 6 6 Choline chlorideUrea 1:2 Zinc chloride (0.95) 0 0 7 Choline chloride Urea 1:2 Zincchloride (0.1) 122 8 8 Choline chloride Urea 1:2 CTAB (0.125) 144 7 9Choline chloride Urea 1:2 Sodium dodecyl sulphate (0.04) 18 9 10 Cholinechloride Urea 1:2 Sodium benzoate (0.8) 19 9 11 Choline chloride Urea1:2 Methyl p-toluenesulphonate (0.1) 64 6 12 Choline chloride Urea 1:2Guanidine isothiocyanate (0.8) 19 3 13 Choline chloride Urea 1:2Ammonium thiosulphate (0.07) 99 6 14 Choline chloride Urea 1:2Dodecyldimethyl(3- 122 5 sulphopropyl)ammonium hydroxide (0.01) 15Choline chloride Trifluoroacetamide 1:2 Sorbitol (5% wt:wt) 274 6 16Choline chloride Trifluoroacetamide 1:2 Sorbitol (10% wt:wt) 272 5 17Choline chloride Trifluoroacetamide 1:2 Sorbitol (15% wt:wt) 254 4 18Choline chloride Trifluoroacetamide 1:2 Sorbitol (20% wt:wt) 96 3 19Choline chloride Trifluoroacetamide 1:2 Sorbitol (25% wt:wt) 15 2 20Choline chloride Trifluoroacetamide 1:2 Xylitol (5% wt:wt) 107 6 21Choline chloride Trifluoroacetamide 1:2 Dithiothreitol (9% wt:wt) 150 722 Choline chloride Trifluoroacetamide 1:2 Zinc chloride (1% wt:wt) 3817 23 Choline chloride Trifluoroacetamide 1:2 Zinc acetate (1% wt:wt) 3704 24 Choline chloride Trifluoroacetamide 1:2 Zinc sulphate•7H2O (0.02%wt:wt) 432 8 25 Choline chloride Trifluoroacetamide 1:2 Zincsulphate•7H2O (0.07% wt:wt) 367 8 26 Choline chloride Trifluoroacetamide1:2 Zinc sulphate•7H2O (0.14% wt:wt) 433 8 27 Choline chlorideTrifluoroacetamide 1:2 Zinc sulphate•7H2O (0.7% wt:wt) 214 8 28 Cholinechloride Trifluoroacetamide 1:2 Zinc sulphate•7H2O (1% wt:wt) 70 8 29Choline chloride Trifluoroacetamide 1:2 Zinc sulphate anhydrous (1%wt:wt) 135 8 30 Choline chloride Trifluoroacetamide 1:2 Zinc EDTA (1%wt:wt) 214 7 31 Choline chloride Trifluoroacetamide 1:2 Zinc gluconate(1% wt:wt) 250 4 32 Choline chloride Trifluoroacetamide 1:2 Silica gel(50% vol:vol) 340 7 34 Choline chloride Trifluoroacetamide 1:2 Molecularsieves 4A (50% 450 7 vol:vol) 33 Choline chloride Trifluoroacetamide 1:2Sodium polyacrylate (50% 308 6 vol:vol) 34 Choline chlorideTrifluoroacetamide 1:2 Healthguards ® (50% vol:vol) 454 7 35 Cholinechloride Trifluoroacetamide 1:2 Ethanol (0.1) 293 6 36 Choline chlorideTrifluoroacetamide 1:2 Ethanol (0.25) 595 4 37 Choline chlorideTrifluoroacetamide 1:2 Ethanol (0.5) 655 3 38 Choline chlorideTrifluoroacetamide 1:2 Ethanol (1) 679 1 39 Trehalose Citric acid 1:1Water (1) 169 2

Qualitative RNA quality scale as follows; 0 (highly degraded) to 10(highest quality). The RNA analysis in Table 1 and 2 was carried out asfollows; ethidium bromide stained, 1% agarose 0.5×TAE gelelectrophoresis followed by visual analysis of a photograph taken underuv light, of the integrity of the 18S and 28S rRNA bands. An RNA samplewith an RNA Quality score of 8 or more has an 18S to 28S rRNA ethidiumbromide staining ratio of 1:2, whilst an RNA sample with an RNA Qualityscore of 5 has an 18S to 28S rRNA staining ratio of approximately 1:1.

4. Mixed Compositions of DES with a Supporting Matrix

In order to improve the physical separation of a DES liquid mixture fromthe stored sample such as a tissue or biopsy, the DES was mixed withvarious supporting matrices. The matrix material is not particularlylimited but it should retain structural strength even when mixed withthe DES and should therefore not dissolve or react. Preferentially thesupporting matrix can be labelled directly with for example a barcode ora shelf-life expiration date by means of a printer or ink pen.

To 3.2 g of PEG (MW8000), agarose, polyacrylate or 3 MM cellulose fibres(Whatmann, UK) was added 10 ml of Choline chloride:Trifluoroacetamide(1:1 mol:mol) and heated with stirring at 100° C. for 30 minutes tohomogenise the mixture which was then poured into a suitable containerto cool.

Such composite mixtures have the advantage of being easier to handlethan liquid DES mixtures and reducing carryover of the DES to theextraction step for example RNA purification. The composite can bepoured, molded, shaped, cut, layered or formed into any number ofcontainers such as 96, 24 or 6-well plates, 1.5 ml microcentrifugetubes, 5, 15 or 50 ml tubes.

5. Stabilisation of RNA in Whole Tissue at Different Temperatures

To either 400 μl of Choline chloride:Urea (1:2 mol:mol) or RNAlater in astandard 2 ml polypropylene microcentrifuge tube was added 10 mg ratliver sample and pre-incubated for 20 minutes at room temperature toallow stabilisation and/or fixation, the intact sample was thenincubated in the DES mixture at −20, 4, 37, 50 or 65° C. for 18 hoursprior to recovery of the intact tissue sample and RNA purification asset out in the following example.

The sample was then added to a fresh tube containing 350 μl of Lysisbuffer RLT, and the RNA extracted from the guanidine homogenised tissueaccording to manufacturer's instructions (RNeasy Mini Kit, Cat. No.74106, Qiagen, Germany). 300 μl portions of the guanidine lysate wasthen used to immediately purify the RNA according to manufacturer'sinstructions and eluted into 20-50 μl of water. RNA yield and qualitywas determined as set out in Example 1, for the DES stabilisationmixture the RNA integrity was superior at all temperatures greater than37° C. and equal at −20 and 4° C. compared with RNAlater. Results areshown in FIG. 3. Lanes 1, 3, 5, 7, 9 Choline chloride:Urea (1:2), Lanes2, 4, 6, 8, 10 RNAlater.

It will be evident to one skilled in the art that Choline chloride:Ureacan be replaced with other DES mixtures such as Cholinechloride:Trifluoroacetamide (1:2 mol:mol).

6. Long Term Stabilisation of RNA in Whole Tissue at 24° C.

To either 400 μl of Choline chloride:Urea (1:2 mol:mol) or RNAlater in astandard 2 ml polypropylene microcentrifuge tube was added 10 mg ratliver sample and pre-incubated for 20 minutes at room temperature toallow stabilisation and/or fixation, the intact sample was thenincubated in Choline chloride:Urea (1:2 mol:mol) at 24° C. for 0-19 daysprior to recovery of the intact tissue sample and RNA purification asset out in the following example.

The sample was added to a fresh tube containing 350 μl of Lysis bufferRLT, and the RNA extracted from the guanidine homogenised tissueaccording to manufacturer's instructions (RNeasy Mini Kit, Cat. No.74106, Qiagen, Germany). 300 μl portions of the guanidine lysate wasthen used to immediately purify the RNA according to manufacturer'sinstructions and eluted into 20 μl of water. RNA yield and quality wasdetermined as set out in Example 1, for the DES stabilisation mixturethe RNA integrity was equal to or superior to RNAlater at all timepoints. Results are shown in FIG. 4. Lanes 1, 3, 5 Choline chloride:Urea(1:2), Lanes 2, 4, 6 RNAlater treated samples.

7. Purification from Different Tissue Types

To either 400 μl of Choline chloride:Urea (1:2 mol:mol) or RNAlater in astandard 2 ml polypropylene microcentrifuge tube was added 10 mgportions of frozen mouse brain (Lanes 3 and 4) or kidney samples (Lanes1, 2, 5, 6) and pre-incubated for 20 minutes at room temperature toallow stabilisation and/or fixation, the intact sample was thenincubated in Choline chloride:Urea (1:2 mol:mol) at 37° C. for 1-7 daysprior to recovery of the intact tissue sample and RNA purification asset out below (FIG. 5A. Lanes 1, 3, 5; Stabilisation with Cholinechloride:Urea (1:2), Lanes 2, 4, 6; stabilisation with RNAlater (Qiagen,France) for either 24 hours (Lanes 1-4) or 7 days (Lanes 5 and 6)).

Alternatively, 10 mg portions of previously frozen mouse tissue wereadded to 400 μl of Choline chloride:Trifluoroacetamide and incubated at37° C. for 18 hours prior to RNA purification (FIG. 5B. Lanes 1, 3, 5,7, 9, 11, 13, 15; stabilisation with Choline chloride:Trifluoroacetamide(1:2), Lanes 2, 4, 6, 8, 9, 10, 12, 14, 16; stabilisation with RNAlater(Qiagen, Germany).

The sample was added to a fresh tube containing 350 μl of Lysis bufferRLT, and the RNA extracted from the guanidine homogenised tissueaccording to manufacturer's instructions (RNeasy Mini Kit, Cat. No.74106, Qiagen, Germany). 300 μl portions of the guanidine lysate wasthen used to immediately purify the RNA according to manufacturer'sinstructions and eluted into 20 μl of water. RNA yield and quality wasdetermined as set out in Example 1, for the DES stabilisation mixturethe RNA integrity was equal to or superior to RNAlater.

8. Purification from Different Amounts of Tissue

To either 400 μl of Choline chloride:Urea (1:2 mol:mol) or RNAlater in astandard 2 ml polypropylene microcentrifuge tube was added either 15 mg(Lanes 1 and 2) or 25 mg of rat liver (Lanes 3 and 4) and pre-incubatedfor 20 minutes at room temperature to allow stabilisation and/orfixation, the intact sample was then incubated in Choline chloride:Ureaat 37° C. for 18 hours prior to recovery of the intact tissue sample andRNA purification as set out below.

The sample was added to a fresh tube containing 350 μl of Lysis bufferRLT, and the RNA extracted from the guanidine homogenised tissueaccording to manufacturer's instructions (RNeasy Mini Kit, Cat. No.74106, Qiagen, Germany). 300 μl portions of the guanidine lysate wasthen used to immediately purify the RNA according to manufacturer'sinstructions and eluted into 20 μl of water.

RNA yield and quality was determined as set out in Example 1, for theDES stabilisation mixture the RNA integrity was equal to or superior toRNAlater at all time points. Results are shown in FIG. 6. Lanes 1 and 3,Choline chloride:Urea (1:2), Lanes 2 and 4, RNAlater treated samples.

It was found that the DES mixture containing Cholinechloride:Trifluoroacetamide (1:2 mol:mol) was significantly moreeffective at stabilising RNA in fresh tissues than Choline chloride:Urea(1:2 mol:mol). However, if the tissue is frozen first at −20° C. or −80°C., Choline chloride:Urea is equally effective as Cholinechloride:Trifluoroacetamide. It is not understood why Cholinechloride:Urea is less effective at stabilising RNA in fresh tissue butit has been found that the addition of 33 mM ZnCl2 or ZnSO4 to theCholine chloride:Urea (2:1 mol:mol) significantly reduces the amount ofRNA degradation occurring when using fresh tissues.

Usefully, tissues such as liver, kidney and muscle treated for at leastone hour with a DES, such as Choline chloride:Trifluoroacetamide (1:2mol:mol) and then frozen at −80° C. are significantly softer thannon-treated tissues allowing the penetration of a biopsy needle. This isparticularly useful when the sample should not be completely thawed butonly a portion removed for further analysis. It should also be notedthat the colour of tissues treated with Cholinechloride:Trifluoroacetamide, notably blood does not significantly changeor fade, whilst untreated or formol treated samples rapidly lose theircolour intensity, with or without freezing and such preservation ofcolour can be an important advantage for correctly analysing biopsyspecimens and cell types in a biopsy specimen.

9. Purification of RNA from DES Stabilised Whole Blood Spiked with HeLaCells

To 400 μl of Choline chloride:Trifluoroacetamide (1:2 mol:mol) was added50 μl of whole human blood spiked with 50 μl of 150,000 HeLa cells andmixed by gentle pipetting, the sample was left to stabilise for 20minutes at room temperature prior to RNA extraction. Either 50 μl(Lane 1) or 100 μl (Lane 2) of this stabilised DES—blood/cell mixturewas mixed with 300 μl of Lysis buffer RLT (RNeasy Mini Kit, Cat. No.74106, Qiagen, Germany) to lyse the cells and extract the RNA,purification was carried out as follows.

The guanidine blood lysate was centrifuged for 60 seconds at 14,000 gand the supernatant transferred to a fresh tube containing 300 μl of 70%ethanol, mixed by pipetting and then transferred to a MinElute spincolumn (RNeasy Micro Kit, Cat. No. 74004, Qiagen, Germany). The MinElutecolumn was washed once with 700 μl of Buffer RW1 then twice with 500 μlof Buffer RPE, centrifuged for 60 seconds to dry the column prior toelution with 20 μl of water according to the manufacturer'sinstructions. The RNA yield was determined by OD260 nm absorbance usinga Nanodrop (ThermoScientific, USA) and loaded and analysed in a 1%agarose, 0.5×TAE gel.

TABLE 3 Yields of RNA derived from HeLa cell spiked blood samples.Sample volume Total RNA Yield 1  50 μl  200 ng 2 100 μl 2400 ng

Results are shown in FIG. 7. It is notable that the silica MinElute spincolumn after passage of the guanidine—blood lysate was not visiblycontaminated with haem. The Cholinechloride:Trifluoroacetamide—guanidine mixture therefore appears toprotect the silica membrane from non-specific contamination.

10. Stabilisation of RNA in Whole Blood Spiked with HeLa Cells UsingCholine Chloride:Trifluoroacetamide

To 1000 μl of Choline chloride:Trifluoroacetamide (1:2 mol:mol) wasadded 200 μl of whole human blood spiked with 50 μl of 1,000,000 HeLacells and mixed by gentle pipetting, the sample was incubated for 18hours at room temperature prior to RNA extraction. Either 100 μl (FIG.8; Lane 1), 150 μl (Lane 2), 200 μl (Lane 3) or 250 μl (Lane 4) of thestabilised DES—blood/cell mixture was mixed with 250 μl of Lysis bufferRLT (RNeasy Mini Kit, Cat. No. 74106, Qiagen, Germany) in order to lysethe cells and extract the RNA, purification was carried out as follows.

The guanidine blood lysate was centrifuged for 60 seconds at 14,000 gand the supernatant transferred to a fresh tube containing 300 μl of 70%ethanol, mixed by pipetting and then transferred to a MinElute spincolumn (RNeasy MinElute, Cat. No. 74204, Qiagen, Germany). The MinElutecolumn was washed once with 700 μl of Buffer RW1 then twice with 500 μlof Buffer RPE, centrifuged for 60 seconds to dry the column prior toelution with 20 μl of water according to the manufacturer'sinstructions. The RNA yield was determined using a Nanodrop (Agilent,USA) and loaded and analysed in a 1% agarose, 0.5×TAE gel. The OD260/280 nm data demonstrates that the RNA is substantially free ofcontaminating protein, whilst the RNA yields suggest that the MinElutecolumns are saturated with RNA and that MinElute columns (RNeasy MiniKit, Cat. No. 74106, Qiagen, Germany) would provide even better yields.

TABLE 4 RNA yields and quality following storage of blood samples for 18hours. Sample volume OD 260/280 nm Total RNA Yield 1 100 μl 2.03  640 ng2 150 μl 2.03 1500 ng 3 200 μl 2.01 1120 ng 4 250 μl 2.09  600 ng

Stabilisation of RNA overnight in whole blood was also demonstrated asfollows. 50,000 HeLa cells were mixed with 50 μl of fresh human wholeblood and then the cells and blood were added to either 400 μl of BufferRLT (Qiagen, Germany) or 400 μl of Choline chloride:Trifluoroacetamide(1:2 mol:mol), and incubated overnight at 24° C., RNA was then purifiedaccording to manufacturer's instructions (RNeasy Mini, Qiagen, Germany).The RNA was significantly protected in whole blood from degradation byCholine chloride:Trifluoroacetamide (1:2 mol:mol) but degraded whenstored overnight in Buffer RLT. Storage of RNA in whole blood, either ina cellular form such as white blood cells or circulating tumour cells,in a sub-cellular form such as exosomes or other microvesicles, orwithin viral particles can be carried out by adding 1:8 or morepreferably, 1:10 of whole blood to Choline chloride:Trifluoroacetamide(1:2 mol:mol). Alternatively 10 mM ZnCl2 or ZnSO4 with or without 20%wt:wt Molecular sieves 4A can be added to the Cholinechloride:Trifluoroacetamide to improve RNA stability further.

FIG. 9 shows stabilisation of RNA in whole blood with either Guanidineor Choline chloride:Trifluoroacetamide (1:2 mol:mol). Storage of samplesovernight at 24° C. in either Buffer RLT (Qiagen, Germany) (Lane 1) or400 μl of Choline chloride:Trifluoroacetamide (1:2 mol:mol) (Lane 2),Total RNA becomes significantly degraded in guanidine but not Cholinechloride:Trifluoroacetamide.

11. Stabilisation of Whole Blood Using a Vacuum Blood Draw Tube

To a 10 ml polyethylene terephthalate (PET) blood collection tube wasadded 7 ml of sterile Choline chloride:Trifluoroacetamide (1:2 mol:mol),and the tube closed with a HEMOGARD™ (Becton Dickinson, USA) or otherappropriate closure and the air partially removed to create a vacuum.Alternatively, the blood collection tube can contain in addition to the7 ml of Choline chloride:Trifluoroacetamide (1:2 mol:mol) ZnSO4 to givea final concentration in the diluted blood sample of either 1 mM, 5 mM,10 mM, 33 mM, 100 mM or 200 mM. As an example of a blood draw tubedevice see FIG. 1, right. Approximately 2 ml of whole venous blood wasdrawn into the tube using a blood collection set (PreAnalytix, Germany)or via filling a regular luer-lock syringe and needle, and transferring2 ml of the contents to the blood-collection tube. Following addition ofthe blood, the tube was inverted 10 times in order to mix the componentsand then incubated for 20 minutes at room-temperature to fix andstabilise the RNA in white blood cells such as T- and B-lymphocytes,monocytes, macrophages (e.g. PBMC), neutrophils, basophil andoesonophils (polymorphonuclear cells), thrombocytes and any bacteria orviruses as set out in the description including HPV, HIV, HCV, HBV,Influenza and coronaviruses implicated in SARS. In general erythrocytesdo not remain intact in this mixture. Other cell types such ascirculating tumour cells can also be stabilised and fixed using thismethod allowing better capture, analysis and storage of the CTC's.

Following storage in the blood collection tube for up to, for example 24hours at 37° C., 3 days at room-temperature, 1 week at 4° C. or 3 monthsat −20° C., the RNA can be extracted from the Cholinechloride:Trifluoroacetamide stabilised blood as follows: The bloodcollection tube was opened and 1 ml of the stabilised sample was removedand mixed with 3 ml of Lysis Buffer RLT, centrifuged for 60 seconds at14,000 g, the supernatant removed and added to an equal volume of 70%ethanol prior to loading in either a RNeasy MinElute (RNeasy Mini Kit,Cat. No. 74106, Qiagen, Germany) or a RNeasy Midi spin column (RNeasyMidi Kit, Cat. No. 75142, Qiagen, Germany) and then the spin column waswashed with Buffers RW1 and RPE and the RNA eluted according to the kitmanufacturers instructions.

12. Measuring the Cell Fixation Properties of DES Mixtures

To each well in a 12-well tissue culture plate was added 20,000 freshlytrypsinised HeLa cells in 1 ml DMEM/5% FBS and the cells then allowed toattach to the plate surface by incubating in an appropriate tissueculture incubator for at least 6 hours at 37° C. The tissue culturemedium was then removed using a vacuum pipette and 400 μl of a DESmixture was added to each well whilst examining any morphologicalchanges of the cells in real-time under a 20× light microscope. Thetissue culture plate was then returned to a 37° C. incubator for 90minutes prior to further microscope examination. Dulbecco's bufferedphosphate saline (DPBS) was used a non-toxic control and results shownin the following table. Cell viability was ascertained by standardTrypan Blue staining.

TABLE 5 Effects of Various Fixatives and Additives on Cell Morphology.Fixative (mol:mol) Effect on cells Cell viability 1 Dulbecco phosphatebuffered saline Unchanged Yes 2 Choline chloride:urea (1:2) Cellscontract and cytoplasm translucent No 3 Choline chloride:Xylitol (1:1)Detach, shrink with crenation No 4 Choline chloride:Sorbitol (1:1)Detach, shrink with crenation No 5 Choline chloride:Trifluoroacetamide(1:2) Unchanged No 6 Choline chloride:Ethylene glycol (1:2) Detach No 7Choline chloride:urea:ZnCl2 (1:2:1) 15% lysis, cells opaque, noshrinkage No 8 Choline chloride:urea:ZnCl2 (10:20:1) Cells opaque and80% reduced size No 9 Choline chloride:urea:CTAB (8:16:1) Homogenouscytoplasm no shrinkage No 10 Choline chloride:urea:SDS (25:50:1)Reasonable morphology, no cell shrinkage No 11 Cholinechloride:urea:Methyl p-toluene Reasonable morphology, opaque, 80%cytoplasmic No sulphonate (3:6:1) condensation 12 Cholinechloride:urea:Sodium benzoate (36:72:1) Cytoplasm hypercondensed, opaqueNo 13 Choline chloride:Guanidine isothiocyanate (2:1) Cells swell andlyse No 14 ZnCl2:Ethylene Glycol (1:4) Cells highly condensed, 50%detached No 15 ZnCl2:Ethylene Glycol:Trifluoroacetamide (1:3:1)Cytoplasm highly condensed, rupture and No heterogenous 16 RNAlaterBlebbing of cytoplasm, otherwise intact and opaque No

TABLE 6 Effects of Various DES Mixtures with or without additives onCell Morphology. Effect on Ratio Additive HeLa Component 1 Component 2(mol:mol) Final % Morphology 1 Choline chloride Trifluoroacetamide  1:1.8 — ++++ 2 Choline chloride Trifluoroacetamide 1:2 — ++++ 3Choline chloride Trifluoroacetamide   1:2.25 — ++ 4 Choline chlorideTrifluoroacetamide   1:2.5 — ++++ 5 Choline chloride Trifluoroacetamide  1:2.75 — ++++ 6 Choline chloride Trifluoroacetamide 1:3 — +++++ 7Choline chloride Trifluoroacetamide 1:2 H2O (17%) +++++ 8 Cholinechloride Trifluoroacetamide 1:2 H2O (13%) +++ 9 Choline chlorideTrifluoroacetamide 1:2 H2O (10%) +++ 10 Choline chlorideTrifluoroacetamide 1:2 H2O (5%) ++++ 11 Choline chlorideTrifluoroacetamide 1:2 H2O (2.5%) +++ 12 Choline chlorideTrifluoroacetamide 1:2 Ethylene glycol (17%) ++ 13 Choline chlorideTrifluoroacetamide 1:2 1,6-Hexanediol (17%) +++ 14 Choline chlorideTrifluoroacetamide 1:2 Ethanol (17%) ++++ 15 Choline chlorideTrifluoroacetamide 1:2 Methanol (17%) +++ 16 Choline chlorideTrifluoroacetamide 1:2 Dimethylformamide (17%) + 17 Choline chlorideTrifluoroacetamide 1:2 Dimethylsulphoxide (17%) +++ 18 Choline chlorideTrifluoroacetamide 1:2 N-Methyl pyrrolidone (17%) +++++ 19 Cholinechloride Trifluoroacetamide 1:2 N-Ethyl pyrrolidone (5%) +++++ 20Choline chloride Trifluoroacetamide 1:2 Ethyleneurea (5%) ++ 21 Cholinechloride Trifluoroacetamide 1:2 Pivalamide (5%) +++ 22 Choline chlorideTrifluoroacetamide 1:2 1,3-Dimethylurea (5%) ++ 23 Choline chlorideTrifluoroacetamide 1:2 N,N′-Dimethylourea (5%) ++ 24 Choline chlorideTrifluoroacetamide 1:2 Isopropanol (17%) ++++ 25 Choline chlorideTrifluoroacetamide 1:2 Butanol (17%) ++++ 26 Choline chlorideTrifluoroacetamide 1:2 Glycerol (17%) ++ 27 Choline chlorideTrifluoroacetamide 1:2 1-Methylimidazole (33%) +++++ 28 Choline chlorideTrifluoroacetamide 1:2 1-Methylimidazole (5%) ++ 29 Choline chlorideTrifluoroacetamide 1:2 1-Ethylimidazole (5%) ++ 30 Choline chlorideTrifluoroacetamide 1:2 1-Benzylimidazole (2.5%) +++++ 31 Cholinechloride Trifluoroacetamide 1:2 1-Benzylimidazole (5%) +++++ 32 Cholinechloride Trifluoroacetamide 1:2 Tetramethylurea (1%) +++++ 33 Cholinechloride Trifluoroacetamide 1:2 Tetramethylurea (5%) +++++ 34 Cholinechloride Trifluoroacetamide 1:2 Ethylene carbonate (33%) ++ 35 Cholinechloride Trifluoroacetamide 1:2 Imidazole 33%) ++++ 36 Choline chlorideTrifluoroacetamide 1:2 Lithium acetate (33%) ++ 37 Choline chlorideTrifluoroacetamide 1:2 4-Formyl morpholine (33%) +++++ 38 Cholinechloride Trifluoroacetamide 1:2 Acetonyl acetone (20%) ++ 39 Cholinechloride Trifluoroacetamide 1:2 Guanidine HCl (3.4%) ++ 40 Cholinechloride Acrylamide 1:2 — ++ 41 Choline chloride 2-Chloroacetamide 1:2 —++ 42 Choline chloride Bistrifluoroacetamide 1:2 — ++++ 43 Cholinechloride 2,2-Difluoropropanamide 1:2 — +++ 44 Choline chloride2,2,2-Trifluorothioacetamide 1:2 — ++ 45 Choline chloride Formamide 1:2— ++ 46 Choline chloride Methanol 1:2 — ++ 47 Choline chloride Ethanol1:2 — ++ 48 Choline chloride Trifluoroacetamide 1:2 Sorbitol (5%) ++ 49Choline chloride Trifluoroacetamide 1:2 Xylitol (5%) ++ 50 Cholinechloride Urea 1:2 — ++ 51 Choline chloride Urea 1:2 Na cacodylate (10%)+++ 52 Choline chloride Urea 1:2 SDS (5%) ++ 53 Choline chloride Urea1:2 Na p-Toluene sulphonic acid (6%) + 54 Choline chloride Urea 1:2Triton TX-45 (12%) + 55 Choline chloride Urea 1:2 Na benzoate (8%) +++56 Choline chloride Urea 1:2 Guanidine isothiocyanate (7%) + 57 Cholinechloride Urea 1:2 Sulpho salicylic acid (10%) + 58 Choline chloride Urea1:2 CTAB (8%) ++ 59 Choline chloride Urea 1:2 Zinc chloride (11%) +++ 60Choline chloride Urea 1:2 Methyl p-toluenesulphonate (25%) ++ 61 ZnCl2Ethylene glycol 1:4 — + 62 ZnCl2 Ethylene 1:3:1 — +glycol:Trifluoroacetamide 63 ZnCl2 Urea   1:3.5 — + 64 ZnCl2Trifluoroacetamide   1:3.5 — ++ 65 Choline chloride Sorbitol 1:1 — ++++66 Choline chloride Guanidine isothiocyanate 2:1 — + 67 Choline chloridePhenylacetic acid 1:2 — + 68 Choline chloride Malonic acid 1:2 — + 69Choline chloride Boric acid   1:1.5 — +++ 70 Acetylcholine chloride Urea1:2 — + 71 Acetylcholine chloride Trifluoroacetamide 1:2 — ++ 72 Cholinebromide Urea 1:2 — + 73 Choline bromide Trifluoroacetamide 1:2 — ++++ 74Beta-methylcholine Trifluoroacetamide 1:2 — + chloride 75 CarnitineTrifluoroacetamide 1:2 — ++ 76 Taurine Trifluoroacetamide 1:2 — + 77Methyltriphenylphosphonium Trifluoroacetamide 1:3 — + bromide 78Grignard Reagent T Trifluoroacetamide 1:2 — +++ 79 ChloroethyltrimethylTrifluoroacetamide 1:2 — +++ ammonium chloride 80 CetyltrimethylammoniumTrifluoroacetamide 1:2 — ++ chloride 81 Tetramethyl ammoniumTrifluoroacetamide 1:2 — + oxide 82 Choline chloride Trichloroacetamide1:2 — ++ 83 Benzyltrimethylammonium Trifluoroacetamide 1:2 — +++chloride 84 Betaine Trifluoroacetamide 1:2 — +++ Effect on HeLa cellmorphology, scale + (worst) to +++++ (best).

13. Cell Fixation with Trifluoroacetamide Containing DES Mixtures

HeLa tissue culture cells were grown under standard tissue cultureconditions in a 24 well tissue culture plate to confluence, the 1 ml ofDMEM/FBS medium was removed and replaced with 0.2-1.0 ml of (A)Dulbecco's phosphate buffered saline (DPBS) or (B) Cholinechloride:Trifluoroacetamide (1:2 mol:mol) and the cells imaged under a50× standard light microscope. Representative fields of cells are shownin FIG. 10, no substantial changes to the cell morphology were seenbetween the DPBS or Choline chloride:Trifluoroacetamide treated cells.As one test to demonstrate that the DES treated cells were fixed, theDPBS or Choline chloride:Trifluoroacetamide was removed and the cellswashed with 2 ml tap water, it was found that, after one hour at roomtemperature, only the DPBS treated cells swelled and then ruptured fromthe osmotic effect of the water, whilst the Cholinechloride:Trifluoroacetamide treated cells remained largely unchanged bythis additional treatment even after 1 month submersion in water at roomtemperature demonstrating that they had indeed been fixed. Furthermore,as proof of the fixation of the cells, they were treated with 1 mL of0.05% Trypsin for one hour at room temperature and it was found that,unlike with DPBS treated cells, there was no effect or visible proteasedegradation of the cells and they remained intact.

The Choline chloride:Trifluoroacetamide (1:2 mol:mol) can be replacedwith Choline chloride:Trifluoroacetamide 1:1, 1:1.5, 1:1:75, 1:2.25,1:2.5, 1:2.75 or 1:3 (mol:mol). Alternatively the Cholinechloride:Trifluoroacetamide can be replaced withBetaine:Trifluoroacetamide (1:2 mol:mol) or Acetylcholinechloride:Trifluoroacetamide (1:2 mol:mol). There is no particularlimitation to the DES mixture for cell fixation but Trifluoroacetamidecontaining mixtures are particularly useful for cell fixation and RNAstabilisation (see Table 1).

Tissue culture cells and tissues can be fixed with Cholinechloride:Trifluoroacetamide (1:2 mol:mol) at different temperatureswithout the cells lysing or becoming distorted. 400 μl portions ofCholine chloride:Trifluoroacetamide were preheated at 37° C., 100° C. or120° C., and with a pre-heated pipette tip added to HeLa cells in a24-well plate. Immediate microscope observation of the cells after theaddition of the hot Choline chloride:Trifluoroacetamide showed that,remarkably, they had a morphology very similar to cells fixed at roomtemperature. The viscosity of hot Choline chloride:Trifluoroacetamide issignificantly less than at room temperature.

Specifically, the cell fixation properties of the deep eutectic solventwere determined and quantified as follows: approximately 2,000 HeLacells were grown on a 25 mm Cellattice™: Micro-Ruled Cell CultureSurface (Micro-ruled cell culture coverslip surface, Cat. No.CLS5-25D-050 Nexcelom Bioscience, USA) placed in a 24-well tissueculture plate and grown overnight in 2 ml of DMEM/10% FBS, the number ofattached cells in a defined area of the grid was counted manually usinga 10× objective microscope lens, the tissue culture medium was thenremoved using an aspirating pipette and replaced with 400 mg of a deepeutectic solvent, incubated for 1 hour at room temperature to allow cellfixation and then the deep eutectic solvent removed with an aspiratingpipette and replaced with 2 ml of distilled water, incubated for 1 hourat room temperature and the number of cells in the same defined area ofthe grid as before treatment counted manually. The percentage ofattached cells remaining in the grid compared with the original numberwas calculated and it was found that at least 75% of the cells wereattached following treatment with Choline chloride:Trifluoroacetamide(1:2 mol:mol). Note the cells should not be grown to confluence as largenumbers of loosely attached dying cells have been found to detach easilyand therefore cause errors in cell counting. It will be evident to oneskilled in the art that the cell fixation properties of other deepeutectic solvents can also be determined using this method.

14. Solubility of Salt Mixtures in Choline Chloride:Urea

It was notably found that Zinc chloride (ZnCl2) could be dissolved inCholine chloride:Urea (1:2) to give a DES mixture of Cholinechloride:Urea:ZnCl2 of 1:2:2 (mol:mol:mol), Guanidine isothiocyanate canbe dissolved in Choline chloride:Urea to give a DES mixture of Cholinechloride:Urea:Guanidine isothiocyanate of 1:2:5 (mol:mol:mol) andAmmonium acetate could be dissolved in Choline chloride:Urea (1:2) togive a DES mixture of Choline chloride:Urea:Ammonium acetate of 1:2:3(mol:mol:mol).

15. RNA Degradation in the Absence of a Stabiliser

In order to determine the rate of RNA degradation in the absence of aDES mixture or other stabiliser, 50 mg pieces of rat liver wereincubated at 20° C. for (Lane 1) 0 min, (Lane 2) 1 min, (Lane 3) 2 min,(Lane 4) 5 min, or (Lane 5) 20 min prior to RNA purification accordingto Example 1. Results are shown in FIG. 10.

It was found that the RNA was noticeably degraded after 5 minutes atroom temperature and significantly degraded after 20 minutes. Thisprovides a method to estimate the maximum amount of time that RNA in atissue can remain intact before it starts to degrade and therefore therapidity and efficacy that the DES fixative can be compared against. Forexample the weight of samples from a tissue with a relatively low rateof RNA degradation such as muscle can be larger than from a tissue witha higher rate such as pancreas.

16. DNA Stabilisation in Animal Tissue Samples

To 400 μl of Choline chloride:Trifluoroacetamide (1:2 mol:mol) in astandard 1.5 ml polypropylene microcentrifuge tube was added 2-25 mg ratliver sample and pre-incubated for 20 minutes at room temperature toallow stabilisation and/or fixation, the sample can then be incubated at−80, −20, 4, 20 or 37, 42 or 55° C. for one hour to several weeks priorto recovery of the tissue sample with forceps followed by RNA and thenDNA purification as set out below.

Briefly, the sample is mechanically lysed in 400 μl of Lysis buffer RLTand the RNA eluted in at least 40 μl of water according tomanufacturer's instructions (RNeasy Mini Kit, Cat. No. 74106, Qiagen,Germany), the silica membrane is then washed with 100 μl of water,centrifuged for 60 seconds at 10,000×g and the flow through discarded,then 100 μl of 10 mM NaOH is added, incubated at 70° C. for 15 minutesto destroy residual RNA and then centrifuged for 60 seconds at 10,000×gand the flow through containing the DNA collected and analysed using a1% agarose gel.

Commercialised DNA purification kits such as the PureLink® (Cat. No.12183018A, Life Technologies, USA) and DNeasy Mini Kit, (DNeasy MiniKit, Cat. No. 69504, Qiagen, Germany) can also be used and there is noparticular limitation to the type of kit or type of tissue that can beused for DNA purification.

The liver sample can be replaced with other tissue and cell types suchas liver, spleen, brain, muscle, heart, oesophagus, testis, ovaries,thymus, kidneys, skin, intestine, pancreas, adrenal glands, lungs, bonemarrow or cells such as COS-7, NIH/3T3, HeLa, 293, and CHO cells or evenliquid samples such as serum, plasma or blood.

It was found that DNA extracted from rat liver samples that had beenfixed and stabilised in 400 μl of Choline chloride:Trifluoroacetamide(1:2) compared with 400 μl of RNAlater at room-temperature hadsignificantly more intact DNA demonstrating the superior stabilisationof DES mixture compared with RNAlater, a product that has beenrecommended to preserve DNA as well as RNA. 1-33 mM ZnSO4 can also beadded to improve DNA stabilisation.

Results are shown in FIG. 12. HeLa cell pellets stabilised in; Cholinechloride:Trifluoroacetamide (1:2 mol:mol) (Lane 1 and 3), RNAlater(Lanes 2 and 3), for either 9 (Lanes 1 and 2) or 15 days (Lanes 3 and 4)at 24° C. The DNA in RNAlater stabilised samples is significantly moredegraded than with Choline chloride:Trifluoroacetamide.

17. Protein Stabilisation in Animal Tissue Samples

To 400 μl of Choline chloride:Trifluoroacetamide (1:2 mol:mol), 10 mMZnSO4.7H2O and 40 mg Molecular sieves 4A in a standard 1.5 mlpolypropylene microcentrifuge tube was added 10 mg of frozen-thawedmouse liver and incubated for either 4, 7 or 18 days at 24° C. Controlmouse liver samples were incubated in 400 μl of PBS for either 0minutes, 36 hours, 6 days or 13 days at 24° C. prior to proteinextraction.

Proteins were extracted by adding 10 volumes of 1× Sample buffer (125 mMTris-HCl pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.001%bromophenol blue) to the liver sample, grinding with a Pellet pestle for30 seconds and then immediately heating the sample for 10 minutes at 70°C., placing the tube on ice for 5 minutes and then centrifuging for 5minutes at 10,000×g prior to protein dosing by the Bradford method(Bio-Rad, France). 30 μg of each protein were mixed with Laemlli bufferand loaded in a standard SDS-7.5% acrylamide gel and electrophoresed for3 hours at 110V. The proteins were then transferred to a Westernblotting PVDF/ECL+ membrane and incubated overnight in TBS (0.1%Tween-20), 5% milk powder at 4° C. with a 1:500 dilution of the primaryantibody anti-α-actin, the membrane was washed three times with TBS(0.1% Tween-20, 5% milk powder) and incubated for 60 minutes at 24° C.with a 1:100 dilution of a HRP labelled mouse anti-IgG secondaryantibody, washing and development with Supersignal West picochemiluminescent kit (Pierce, France).

Results are shown in FIG. 13. Mouse liver stabilised in; PBS (Lanes 2-4)or Choline chloride:Trifluoroacetamide (1:2 mol:mol) (Lanes 5-7), foreither 0 minutes (Lane 1), 36 hours (Lane 2), 6 days (Lane 3), 13 days(Lane 4), 4 days (Lane 5), 7 days (Lane 6) or 18 days (Lane 7) at 24° C.The IgG and actin proteins in PBS stored samples are significantly moredegraded than with Choline chloride:Trifluoroacetamide.

18. Cell Fixation with Choline Chloride:Trifluoroacetamide (1:2 mol:mol)for Immunohistochemistry (IHC)

HeLa cells were grown to 20% cell density on 13 mm glass coverslips in a24-well tissue culture plate, the DMEM growth media was removed with avacuum pipette, the edge dabbed dry with a tissue and the coversliptransferred to a 12 well plate and 600 μl of Cholinechloride:Trifluoroacetamide (1:2 mol:mol) added directly onto the coverslip and left for 60 minutes on a rocking platform at room temperatureto allow fixation. The coverslip and cells were then removed from thefixative, excess fixative removed with a vacuum pipette and dabbing witha paper tissue, and washed for 4×5 minutes with 2 ml of PBS. The cellswere blocked with 2 ml of PBS/1% BSA on a rocking platform, then asuitable dilution, such as 1:100, of the primary antibody was added andleft over night at 4° C. The cells were then washed in 3×2 ml of PBS/1%BSA for 5 minutes each, and a suitable dilution, such as 1:1000 ofAlexafluor 488 goat anti-mouse IgG1 (Life Technologies, UK), of thesecondary labelled antibody was added and incubated in the dark for 30minutes at room temperature. The cells were washed in 3×2 ml of PBS/1%BSA and then 3×2 ml of PBS and then briefly rinsed in water prior tomounting with Vectashield/DAPI (Vector Labs, UK) and observation with asuitable microscope.

Alternatively, the addition of 10 mM ZnSO4, ZnCl2, 5% (vol:vol)N-Ethylpyrrolidone, 5-10% of an aqueous solution such as water, PBS orDMEM, 2.5% (vol:vol) 1-Benzylimidazole or 1% (vol:vol) Tetramethylureainto the Choline chloride:Trifluoroacetamide (1:2 mol:mol) can be madeprior to cell fixation to improve the immunohistochemistry results.

19. Mammalian Tissue Fixation with Choline Chloride:Trifluoroacetamide(1:2 mol:mol) for Staining or Immunohistochemistry (IHC)

Freshly dissected mouse tissue pieces such as liver, kidney, lung,brain, smooth, skeletal or cardiac muscle, spleen, thymus, salivarygland, uterus, testis, skin, eye, tongue, oesophagus, stomach,intestine, pancreas, adrenal glands, gall bladder, were added to 10volumes of Choline chloride:Trifluoroacetamide (1:2 mol:mol) andincubated between 4° C. or room temperature for at least one hour toallow penetration and tissue fixation to occur. Longer incubationperiods of greater than one hour are also compatible, for example 4, 8,15, 24 or 72 hours. The tissue sample can also be frozen and stored inthe Choline chloride:Trifluoroacetamide (1:2 mol:mol) mixture untilneeded. The required time for tissue fixation will depend on a number offactors including the tissue type, size, density, fat content, shape,surface area and the fixative type. Determining the minimum timenecessary for fixation for a particular tissue can be carried out mostsimply by incubating the tissue for different lengths of time and thenobserving how the tissue performs during microtome sectioning;insufficient fixation time would be detected by the tissue tearingduring the passage of the microtome blade. Sufficient fixation timeleads to a robust sample for microtome sectioning but also RNAstabilisation.

Following fixation in Choline chloride:Trifluoroacetamide (1:2 mol:mol)the tissue is rinsed once briefly in 10 volumes of PBS prior todehydration in 70% ethanol for 45 minutes, 80% ethanol for 45 minutes,twice in 100% ethanol for 30 minutes, twice in toluene for 30 minutesprior to embedding in paraffin (melting point 56-58° C.) at 65° C. and100° C. for 1 hour each. The paraffin block containing the fixed tissueis allowed to cool to room temperature prior to microtoming according tostandard protocols identical to those used for formaldehyde fixedtissues. Detailed methods are set out in Al-Mulla and Gohlmann (2011)Formalin-Fixed Paraffin-Embedded Tissues: Methods and Protocols (Methodsin Molecular Biology). Toluene can be replaced with xylene or Histosolif required.

The addition of 1-33 mM, preferably 10-33 mM of Zinc salts such as Zincchloride, Zinc sulphate or Zinc citrate to the Cholinechloride:Trifluoroacetamide improves the rate of penetration andfixation of the tissue by the fixative, whilst the additional presenceof Molecular sieves Type 4A improves RNA stabilisation in the sample.

Tissue section staining with Haemotoxylin and Eosin was according tostandard and well known methods.

20. HeLa Cell RNA and DNA Stabilisation with CholineChloride:Trifluoroacetamide Following Paraffin Embedding

HeLa cell pellets (one million cells) were added to 400 mg of Cholinechloride:Trifluoroacetamide (1:2 mol:mol) containing 10 mM ZnCl2 andfixed for 60 minutes at room temperature. The fixed cells were eitherprocessed immediately or a standard paraffin embedding protocol wasfollowed; (i) 30 minutes immersion in 1 ml of 100% ethanol, (ii) 15minutes with 1 ml Toluene, then either a (iii) 15 or (iv) 60 minuteinfiltration with 1 ml paraffin at 55° C. RNA and DNA was subsequentlypurified (RNeasy, Qiagen, Germany) and the RIN determined (AgilentBioanalyser 2100). The RIN of the HeLa cell RNA decreased from 9.6 (Lane1, positive control) with no fixation, to 8.6 (Lane 6) followingfixation, dehydration and paraffin embedding, demonstrating thatalthough some RNA degradation did occur during processing, the overallamount was very acceptable. It was also found that Cholinechloride:Trifluoroacetamide fixation resulted in far less RNAdegradation than with formaldehyde treated samples (data not shown). Theintegrity of the DNA samples did not visibly change demonstrating thatDNA is also stabilised during fixation. Results shown in FIG. 14.

21. Mouse Liver and Kidney Tissue RNA and DNA Stabilisation with CholineChloride:Trifluoroacetamide Following Paraffin Embedding

10 mg pieces of mouse liver or kidney were added to either 400 μl ofCholine chloride:Trifluoroacetamide (1:2 mol:mol) containing both 10 mMZnSO4 and Molecular sieves 4A (3% (wt:wt), or to 400 μl of PBS andincubated for 64 hours at either 4° C. or 24° C. The tissue samples werethen processed at as follows; 60 min in 70% ethanol, 60 min in 80%ethanol, 60 min in 95% ethanol, two times 30 min in 100% ethanol, 60 minin 100% ethanol, two times 30 min in toluene, 60 min in 100% toluene, 2hours in paraffin at 55° C., 5 hours in paraffin at 55° C., the sampleembedded in the paraffin was then frozen for approximately 2 weeks at−80° C. RNA and DNA was subsequently purified by first removing theembedded tissue from the paraffin block using a scalpel, and then directlysis in 400 μl of buffer RLT using an RNeasy mini kit (Qiagen, Germany)and the RIN determined using an RNA 6000 Nano total RNA kit (AgilentBioanalyser 2100, USA).

Results are shown in FIG. 15. It was found that for both the liver(Lanes 1-4) and kidney (Lanes 5-8) samples, RNA integrity wassignificantly better following Choline chloride:Trifluoroacetamide,ZnSO4 and Molecular sieves treatment (Lanes 1, 2, 5, 6) compared withPBS (Lanes 3, 4, 7, 8). As an example, the RIN values are shown in FIG.14 and were found to decrease from 7.5 to 2.4 comparing Cholinechloride:Trifluoroacetamide, ZnSO4 and Molecular sieves (Lane 1) withPBS (Lane 3) at 24° C., the DNA quality was also found to besignificantly better following Choline chloride:Trifluoroacetamide,ZnSO4 and Molecular sieves treatment.

22. HeLa Cell RNA Stabilisation with CholineChloride:Trifluoroacetamide, Zinc Sulphate and Molecular Sieves

A comparison was made of the RNA stabilisation effect of adding variousZinc salts and Molecular sieves to Choline chloride:Trifluoroacetamide(1:2 mol:mol) in stored biological samples with or without added water.A freshly centrifuged pellet of one million HeLa cells was used as asource of RNA, and 400 μl of Choline chloride:Trifluoroacetamide (1:2mol:mol) was added to each pellet, then water at either a finalconcentration of 10 or 15% (vol:vol) was added in the presence orabsence of 33 mM Zinc sulphate and 33% (wt:wt) Molecular sieves Type 4Aas set out in the table. The samples were stored for 18 hours at 37° C.prior to RNA purification using silica spin columns (Invitek, Germany)and RNA Integrity Number (RIN) determination using an AgilentBioanalyser 2100 according to manufacturer's instructions. Whilst theaddition of water to the HeLa cell pellet/Cholinechloride:Trifluoroacetamide markedly reduces the integrity of the RNA,the addition of Zinc sulphate, or more preferably Zinc sulphate andMolecular sieves Type 4A can substantially reduce the amount of RNAdegradation when water is present as indicated by an increase in the RINnumber. This is particularly useful means to improve sample analytequality when substantial amounts of water (for example more than 10%final concentration in the stabilising solution) are present such aswith larger tissue samples, blood, serum, plasma or plant material. Someimprovement may also be obtained with samples containing less than 10%water when extended sample storage is necessary.

It has been found that Zinc sulphate at 1-33 mM, preferably 10 mM (finalconcentration) is slightly more effective at reducing RNA degradationthan Zinc chloride or Zinc EDTA, but significantly more effective thanZinc gluconate, Zinc acetate or Zinc p-Toluene sulphonate (Table 2).

TABLE 7 RIN Scores for RNA extracted from HeLa cell pellets. DES Mixture(mol:mol) Additive RIN Score 1 Choline chloride:Trifluoroacetamide (1:2)— 8.4 2 Choline chloride:Trifluoroacetamide (1:2) 10% water 6.2 3Choline chloride:Trifluoroacetamide (1:2) 10% water + 33 mM ZnSO4 7.3 4Choline chloride:Trifluoroacetamide (1:2) 10% water + 33 mM ZnSO4 + 33%8.2 Molecular sieves 5 Choline chloride:Trifluoroacetamide (1:2) 15%water 2.9 6 Choline chloride:Trifluoroacetamide (1:2) 15% water + 33 mMZnSO4 5.3

23. HeLa Cell RNA Stabilisation with Choline Chloride:Trifluoroacetamidewith Organic Additives

A comparison was made of the RNA stabilisation effect of addingN-Ethylpyrrolidone or Tetramethylurea to Cholinechloride:Trifluoroacetamide (1:2 mol:mol) in stored biological sampleswith or without added water. A freshly centrifuged pellet of one millionHeLa cells was used as a source of RNA, and 400 μl of Cholinechloride:Trifluoroacetamide (1:2 mol:mol) was added to each pellet, inthe presence or absence of 2.5, 5%, 10% or 20% (vol:vol)N-Ethylpyrrolidone, 5% or 20% (vol:vol) Tetramethylurea as set out inthe table. The samples were stored for 20 days at 24° C. prior to RNApurification using silica spin columns (InviTrap Spin Universal RNA MiniKit Cat. No. 1060100200 Stratec Molecular, Germany) and RNA IntegrityNumber (RIN) determination using an Agilent Bioanalyser 2100 accordingto manufacturer's instructions. It was found that bothN-Ethylpyrrolidone and Tetramethyurea improved RNA quality in the HeLacell pellet following prolonged storage compared with Cholinechloride:Trifluoroacetamide alone.

TABLE 8 RNA Yields and Quality with N-Ethylpyrrolidone andTetramethylurea. DES Mixture Additive RNA Yield ng/ul RNA QualityCholine chloride:Trifluoroacetamide (1:2) — 219 7 Cholinechloride:Trifluoroacetamide (1:2) 2.5% N-Ethylpyrrolidone 92 7 Cholinechloride:Trifluoroacetamide (1:2) 5% N-Ethylpyrrolidone 87 8 Cholinechloride:Trifluoroacetamide (1:2) 10% N-Ethylpyrrolidone 54 9 Cholinechloride:Trifluoroacetamide (1:2) 20% N-Ethylpyrrolidone 139 9 Cholinechloride:Trifluoroacetamide (1:2) 5% Tetramethylurea 194 8 Cholinechloride:Trifluoroacetamide (1:2) 20% Tetramethylurea 211 7

Qualitative RNA quality scale as follows; 0 (highly degraded) to 10(highest quality). The RNA analysis in Table 1 and 2 was carried out asfollows; ethidium bromide stained, 1% agarose 0.5×TAE gelelectrophoresis followed by visual analysis of a photograph taken underuv light, of the integrity of the 18S and 28S rRNA bands. An RNA samplewith an RNA Quality score of 8 or more has an 18S to 28S rRNA ethidiumbromide staining ratio of 1:2, whilst an RNA sample with an RNA Qualityscore of 5 has an 18S to 28S rRNA staining ratio of approximately 1:1.

24. Using Various Quaternary Ammonium Salts and Hydrogen Bond Donors

A room temperature (24° C.) DES liquid could not be prepared from mixingCholine chloride with any of Proline, Oxamide, Pivalamide,1-Ethyl-2-pyrrol, 4-Formyl morpholine, Acetonyl acetone, Ethylenecarbonate, Tetramethyl urea, N-Ethylimidazole, 1-Benzylimidazole and/or1,3-Dimethyl-2-imidazolidone, in a 1:2 mol:mol proportion. The followingammonium salts were also not capable of forming room temperature DESliquids; Ammonium phosphate and Ammonium acetate. Both Ammonium sulphateand Ammonium chloride could partially form, at 100° C. but not at 24°C., a liquid in a 1:2 mol:mol ratio with Guanidine isothiocyanate,Sorbitol and/or Xylitol.

TABLE 9 Two component mixtures using a variety of quaternary ammoniumsalts. Component 1 Component 2 Ratio (mol:mol) Liquid at 100° C. Liquidat 24° C. 1 Choline bromide Trifluoroacetamide 1:2 Yes Partial 2 Cholinechloride Trifluoroacetamide 1:2 Yes Yes 3 Choline iodideTrifluoroacetamide 1:2 Yes No 4 Choline dihydrogen citrateTrifluoroacetamide 1:2 Yes No 5 Choline bitartrate Trifluoroacetamide1:2 Yes No 6 Betaine Trifluoroacetamide 1:2 Yes Yes 7 Ammonium sulphateTrifluoroacetamide 1:2 No No 8 Ammonium sulphate Guanidineisothiocyanate 2:1 No No 9 Ammonium sulphate Guanidine isothiocyanate1:2 Partial No 10 Ammonium sulphate Xylitol 1:2 Partial No 11 Ammoniumsulphate Sorbitol 1:2 Partial No 12 Ammonium chloride Guanidineisothiocyanate 1:2 Partial No 13 Ammonium chloride Xylitol 1:2 Yes No 14Ammonium chloride Sorbitol 1:2 Yes No 15 Ammonium sulphateTrifluoroacetamide 1:2 No No

25. Stabilisation of RNA in Drosophila melanogaster Embryos

10 mg of D. melanogaster embryos (0-24 hours) were mixed with either 400μl of Choline chloride:Trifluoroacetamide (1:2 mol:mol) (Lanes 1-3) orRNAlater (Lanes 4-6) and incubated at 37° C. for either 12 hours (Lanes1, 4), 2 days (Lanes 2, 5) or 45 days (Lanes 3, 6) prior to RNApurification (RNeasy Mini Kit, Cat. No. 74106, Qiagen, Germany). Thequality of RNA is shown in FIG. 16, the Cholinechloride:Trifluoroacetamide stabilised RNA was significantly better thanthat of RNAlater.

26. Stabilisation of RNA in Allium cepa Leaf Shoots

10 mg of A. cepa leaf shoots were mixed with either 400 μl of Cholinechloride:Trifluoroacetamide (1:2 mol:mol) (Lanes 1-3) or RNAlater(Qiagen, Germany) (Lanes 4-6) and incubated at 22° C. for either 18hours (Lanes 1, 4), 3 days (Lanes 2, 5) or 9 days (Lanes 3, 6) prior toRNA purification (RNeasy Mini Kit, Cat. No. 74106, Germany). The qualityof RNA is shown in FIG. 17, the Choline chloride:Trifluoroacetamidestabilised RNA was significantly better than that of RNAlater.

27. In Situ Hybridisation Applications Following DES Stabilisation

Tissue samples were prepared and paraffin embedded as set out in Example21 using Choline chloride:Trifluoroacetamide (1:2 mol:mol) and 10 mMZnSO4 with a fixation time of 1-24 hour at 4° C. The tissue samples werethen processed at as follows; 60 min in 70% ethanol, 60 min in 80%ethanol, 60 min in 95% ethanol, two times 30 min in 100% ethanol, 60 minin 100% ethanol, two times 30 min in toluene, 60 min in 100% toluene, 2hours in paraffin at 55° C. then 5 hours in paraffin at 55° C. Followingmicrotome preparation of the paraffin-tissue slices (3-12 μm thick), theparaffin was removed using xylene for 10 minutes at room temperature,the tissue slices were then hydrated by incubating in 100% ethanol, 70%ethanol, 50% ethanol, 25% ethanol and then water for 5 minutes each. Thetissue sections can then be proteinase K (10 μg/ml) treated for 5minutes at room temperature before rinsing in PBS and pre-hybridisationin 1 ml of buffer containing 500 μl ultra-pure 50% formamide, 250 μl of20×SSC, 50 μl of 10 μg/μl yeast t-RNA and 20 μl of 50×Denhardt'ssolution and then hybridization with an appropriate chromogenic orfluorescently labelled probe. Protocols for in situ hybridisation arewell known and described by J. M. Bridger and K Morris (2010), inFluorescence in situ Hybridization (FISH): Protocols and Applications(Methods in Molecular Biology) and Summersgill et al., (2007) NatureProtoc. 3:220-234.

28. Preparation of Cells for Flow Cytometry

Approximately 500,000 tissue culture cells such as HeLa, MCF-7, NC160,PC3, Vero, GH3, MC3T3, ZF4 or IMR-90, if growing on a solid surface werefirst lightly trypsinised to detach them, mixed with 10 ml of EMEM/10%FBS and centrifuged in a 15 ml tube for 10 minutes at 900×g (24° C.).The cell pellet was then resuspended in 100 μl of DPBS buffer andimmediately mixed with 1 ml of Choline chloride:Trifluoroacetamide (1:2mol:mol) and gently pipetted with a 10 ml pipette to thoroughly mix. Thecells were left to fix for 1-24 hours at either 4° C. or 24° C., then 14ml of DPBS was added, the tube contents mixed by gentle inversion andcentrifuged for 10 minutes at 900×g and the cell pellet gentlyresuspended in 100 μl of DPBS and the nuclei stained by adding 1 ml DAPI(3 μM) in staining buffer (100 mM Tris, pH 7.4, 150 mM NaCl, 1 mM CaCl₂,0.5 mM MgCl₂, 0.1% Nonidet P-40) for 15 minutes (24° C.). The stainedand fixed cells can then be used for flow cytometry. It was found thatthe Choline chloride:Trifluoroacetamide fixed cells were mono-dispersedand could be sorted into the various stages of the cell cycle accordingto their fluorescence.

29. Two-Step Treatment of Biological Samples

10 mg pieces of mouse tissue were added to either 400 μl of Cholinechloride:Trifluoroacetamide (1:2 mol:mol) containing both 10 mM ZnSO4and Molecular sieves 4A (3% (wt:wt), were incubated at 24° C. for 1hour, the tissue was then removed, briefly dabbed with a paper towel toremove excess stabilizer before subsequent immersion in, for example,400 μl of either Choline chloride: Trifluoroacetamide (1:2 mol:mol),Choline chloride:Urea (1:2 mol:mol), Choline chloride:Sorbitol (1:2mol:mol), Betaine chloride:Trifluoacetamide (1:2 mol:mol) or 4%paraformaldehyde and then incubated and stored for at least one hour butpreferably overnight. Alternatively, any one of a number of DES mixturesas set out in this application can serve as the first stabilising orfixation solution followed by a second stabilising or fixation solution.As one more example, tissue fixation can first be carried out with forexample 4% paraformaldehyde for one hour at room temperature and thenthe tissue transferred to 400 μl of Choline chloride:Trifluoroacetamide(1:2 mol:mol) containing both 10 mM ZnSO4 and Molecular sieves 4A (3%(wt:wt). This two-step procedure provides a means by which, for example,the optimum stabilizer for cell morphology can subsequently be combinedwith the optimum stabilizer for RNA, DNA and proteins. It also providesa means by which the water content originating from the biologicalsample can be reduced by changing the original stabilising mixture. Itwill be evident to one skilled in the art that there are manycombinations of the first and second mixtures and that the mostappropriate choices will have to be determined at least in part byempirical means such as quality of H&E stained tissue sections and RNAquality. It should also be noted that the stabilization and fixationmixtures used can either be liquids or solids.

30. Compatibility of DES Mixtures with Guanidine and Phenol PurificationReagents

Advantageously, Choline chloride:Trifluoroacetamide (1:2 mol:mol) iscompletely soluble and compatible with both guanidine thiocyanate or HClbased virus, cell and tissue lysis buffers such as those found in theseRNA purification kits; RNeasy Mini, (Qiagen, Germany), PureLink™ (LifeTechnologies, USA), MagNA Pure LC RNA Isolation Kit III, High Pure RNATissue Kit and RNA Micro Kit Amplicor HCV (Roche Applied Science, USA),NucleoSpin® Multi-8 Virus RAV (Macherey Nagel, Germany), TEMPUS™ BloodRNA Tube (Applied Biosystems, USA), SV RNA Kit and PureYield™ Kit(Promega, USA), ToTALLY RNA™ Kit (Ambion, USA), GenElute™ MammalianTotal RNA Purification (Sigma-Aldrich, USA), PAXgene™ Blood RNA Kit(PreAnalytix, Germany) and phenol based purification reagents such asTRIzol (Life Technologies, USA) allowing Cholinechloride:Trifluoroacetamide (1:2 mol:mol) stabilized samples to bedirectly mixed with guanidine or phenol purification reagents withoutneeding to separate the sample from the Cholinechloride:Trifluoroacetamide. This can be advantageous when, for example,it is not practical to separate a tissue sample that has penetrated bythe fixative, or when individual cells such as tissue culture cells,blood or CTC's are mixed with a much larger volume of fixative and canbe difficult or impossible to separate by centrifugation. As a point ofreference, mammalian cells in RNAlater (Qiagen, Germany) cannot bepelleted by centrifugation or the RNA purified by mixing the cell plusRNAlater with guanidine lysis buffers as the RNA yields dropdramatically.

As one example, it has been found that RNA containing samples containingas little as 6% (Sample 5, Table 10) or less of Buffer RLT in Cholinechloride:Trifluoroacetamide (1:2 mol:mol), can, on mixing with onevolume of 70% ethanol be used to effectively bind RNA to a silica spincolumn membrane (RNeasy mini, Qiagen, Germany) with excellent yield andpurity as set out in Table 10. A mouse liver lysate was prepared bylysing 100 mg of liver in 1 ml of Buffer RLT, then 20 μl portions of thelysate were added to Buffer RLT and then the Cholinechloride:Trifluoroacetamide, before mixing with 70% ethanol as shown inTable 10 and binding to a RNeasy mini spin column. The RNA was thenpurified according to manufacturer's instructions (RNeasy mini, Qiagen,Germany) with an elution volume of 50 μl water. The RNA yields andpurity were determined using a Nanodrop ND-1000. It was surprisinglyfound that not only did Choline chloride:Trifluoroacetamide allow thechaotropic activity of guandine to function to lyse the sample, but ithad no effect on the RNA binding to the silica spin column membrane sothat yields were either not effected or slightly increased.

Furthermore, Choline chloride:Trifluoroacetamide can replace theotherwise essential RNA binding function of 70% ethanol when added tothe guanidine lysate (20 μl), the standard manufacturer's protocol(RNeasy Mini, Qiagen, Germany), and as shown in Table 11, requires theaddition of one volume of 70% ethanol to the lysate to allow the RNA tobind to the silica membrane. If 70% ethanol is not added to the lysatethen RNA cannot bind to the silica membrane, however, and if the samplecontains Choline chloride:Trifluoroacetamide then the RNA can bind evenin the absence of ethanol, this provides a means to reduce the number ofsteps and improve the RNA purification procedure of, for example theRNeasy kit without the need to use flammable liquids. It should be notedthat neither Choline chloride nor Choline chloride:Urea dissolved inBuffer RLT (1:1 wt:wt) have this property, whilst Trifluoroacetamidealone dissolved in Buffer RLT (1:1 wt:wt) led to only 15% of the RNAyield compared with Choline chloride:Trifluoroacetamide dissolved inBuffer RLT (1:1 wt:wt). It was also discovered that a 1:1 mixture ofBuffer RLT:(Choline chloride:Trifluoroacetamide (1:2 mol:mol) had verygood HeLa cell lysis activity and could be used as a standalone lysisand silica membrane binding buffer, in the absence of 70% ethanol, RNAyields with this novel mixture were significantly better than withBuffer RLT alone.

Surprisingly it was found that a HeLa cell lysate prepared in 200 μl ofa 1:1 mixture of Buffer RLT:(Choline chloride:Trifluoroacetamide (1:2mol:mol), when heated at 65° C. for 10 minutes followed by the additionof 1 volume of 70% ethanol and binding to a silica spin column (RNeasyMini, Qiagen, Germany) according to manufacturer's instructions resultedin the exclusive purification of small RNA (miRNA, tRNA and 5S rRNA). Ifthe heating step was omitted total RNA was purified including the 18 and28S rRNA species, heating therefore offers a novel method to selectivelypurify small RNA from a cell lysate. Replacing Trifluoroacetamide withUrea in the Lysis mixture and then heating resulted in extreme RNAdegradation, as did heating the lysate in the absence of Cholinechloride:Trifluoroacetamide.

TABLE 10 RNA yields from guanidine/Choline chloride:Trifluoroacetamidemixtures. Volume RLT Volume Choline Volume 70% RNA yield (guanidine)chloride:Trifluoroacetamide Ethanol OD 260/280 nm ng/ul 1 350 μl  0 μl350 μl 2.05 171 2 150 μl 170 μl 350 μl 2.25 192 3 100 μl 220 μl 350 μl2.22 203 4  50 μl 270 μl 350 μl 2.2 198 5  0 μl 330 μl 350 μl 1.53 200 6170 μl 170 μl  0 μl 2.21 52

TABLE 11 RNA yields from Guanidine/Choline chloride:Trifluoroacetamidemixtures in the absence of ethanol for binding. Volume RLT Volume 70%RNA yield (guanidine) Volume and DES Type Ethanol OD 260/280 nm ng/ul 1330 μl 0 μl Choline 0 μl 1.98 3 chloride:Trifluoroacetamide 2 230 μl 90μl Choline 0 μl 1.84 6.5 chloride:Trifluoroacetamide 3 150 μl 170 μlCholine 0 μl 2.04 143 chloride:Trifluoroacetamide 4 100 μl 220 μlCholine 0 μl 2.04 132 chloride:Trifluoroacetamide 5  50 μl 270 μlCholine 0 μl 2.05 192 chloride:Trifluoroacetamide 6  0 μl 330 μl Choline0 μl 2.03 247 chloride:Trifluoroacetamide 7 150 μl 170 μl Cholinechloride:Urea 0 μl 2.14 9

31. Stabilisation of Total RNA in Bacteria

300 μl of Choline chloride:Trifluoroacetamide (1:2 mol:mol) was added toa 10 mg pellet of Escherischia coli DH5cc and incubated at 22° C. for 18hours, then either the DES liquid was removed and 400 μl of Buffer RLTadded to the pellet, or 400 μl of Buffer RLT was added directly to thepellet and DES liquid, the tube vortexed for 20 seconds, then brieflysonicated to rupture the cells and RNA purification continued using aRNeasy Mini kit according to manufacturer's instructions (Qiagen,Germany). It was found that the integrity of the 16 and 23S rRNA wasunchanged compared with RNA extracted from a fresh bacterial pellet.Alternatively ZnSO4 can be added to the Cholinechloride:Trifluoroacetamide (1:2 mol:mol) to give a final concentrationof 1-33 mM, preferably 33 mM and 10% (wt:wt) Molecular sieves can alsobe optionally added to improve stabilisation.

32. Multi-Component DES Mixtures

It has been found that a RNA stabilising DES mixture can be simplyprepared by mixing more than two components together such asBetaine:Choline chloride:Trifluoroacetamide (0.5:0.5:2 mol:mol:mol)instead of either Betaine:Trifluoroacetamide (1:2 mol:mol) or Cholinechloride:Trifluoroacetamide (1:2 mol:mol). Alternatively, novel DESmixtures can be made from, for example Cholinechloride:Urea:Trifluoroacetamide (1:1:1 mol:mol:mol) orBetaine:Urea:Trifluoroacetamide (1:1:1 mol:mol:mol) or evenBetaine:Choline chloride:Urea:Trifluoroacetamide (0.5:0.5:1:1mol:mol:mol:mol). Such three or more component DES mixtures can haveinteresting novel properties such as reduced viscosity, improvedshelf-life, improved nucleic acid stability or cell fixation propertiesbased on the interactions and properties of all the components togetherin a single DES mixture.

As one example, to a HeLa pellet (500,000 cells) was added 400 mg ofeither Choline chloride; Trifluoroacetamide (1:2 mol:mol),Betaine:Choline chloride:Trifluoroacetamide (0.5:0.5:2 mol:mol:mol) orBetaine:Trifluoroacetamide (1:2 mol:mol) each containing 10 mM ZnSO4 andincubated overnight at 37° C. followed by RNA and DNA purification usinga RNeasy Mini kit (Qiagen, Germany) and determination of the RIN(Agilent Bioanalyser 2100, USA).

It will be evident to one skilled in the art that many such DES mixturesare possible, with variable components and molar concentrations and themost appropriate mixture for the application will need to be determinedempirically.

TABLE 12 Comparison of RNA, DNA yields and RNA Integrity Number (RIN) ofthree different DES mixtures on HeLa cells incubated overnight at 37° C.RNA DNA DES Mixture (including 10 mM ZnSO4) ng/ul ng/ul RIN 1 Cholinechloride:Trifluoroacetamide 251 36 9.3 (1:2 mol:mol), 2 Betaine:Cholinechloride:Trifluoroacetamide 229 42 9.5 (0.5:0.5:2 mol:mol:mol) 3Betaine:Trifluoroacetamide 182 30 9.4 (1:2 mol:mol)

33. Aqueous Mixtures of DES for Cell Fixation

It has been found that aqueous dilutions of Cholinechloride:Trifluoroacetamide (1:2 mol:mol) are capable of fixing tissueculture cells and tissues. DMEM tissue culture medium (LifeTechnologies, France) was added to a solution of Cholinechloride:Trifluoroacetamide to give a final concentration of 0, 6, 12,21 or 50% DMEM, 400 μl portions of the mixture was then added to HeLatissue culture cells in a 24-well plate and observed with a microscope.It was discovered that whilst all the mixtures could fix the cellswithout hypo- or hyper-tonic effects on the cells, Cholinechloride:Trifluoroacetamide containing 6% DMEM led to the best qualitycell morphology, superior even to pure Cholinechloride:Trifluoroacetamide. It should be noted that dilutions ofCholine chloride:Trifluoroacetamide with greater than 15% water cancause the cell membrane to form microdroplets and then be lost from thecell, the cytolasm of which remains intact. Aqueous dilutions of a DESprovides a simple means to reduce the viscosity and cost as well aspotentially improving the cell fixation properties, however the presenceof water has a deleterious effect on RNA stability. It will be apparentto one skilled in the art that a large number of different aqueoussolutions such as water, PBS, DPBS, sugar solutions or DMEM withdifferent DES's are capable of being mixed and that the effect on cellfixation and biomolecule stability may have to be tested empirically.

34. Anti-Bacterial Activity of DES Mixtures

Pellets of 1×10⁹ E. coli DH5a cells were treated with 90 μl of anaqueous dilution of Choline chloride:Trifluoroacetamide (1:2 mol:mol) togive a final concentration of either 90%, 9% or 0.9%, for 25 minutes atroom temperature and then plated onto an agar plate and incubatedovernight at 37° C. to allow colony growth. It was found the 90% Cholinechloride:Trifluoroacetamide but not the 9% or 0.9% dilutions stopped allbacterial growth and colony formation. Cholinechloride:Trifluoroacetamide therefore appears to be a have a powerfulanti-bacterial activity, and it will be evident to one skilled in theart that longer treatment periods or different DES mixtures may lead toan even stronger anti-effect. Advantageously bacterial growth would beexpected to be inhibited in tissue samples stored in Cholinechloride:Trifluoroacetamide stopping spoilage.

35. In Situ Preparation of a DES Liquid

Whilst it is usually convenient to prepare a DES mixture in advance ofits use for fixation and stabilisation, an alternative is to add the twoor more components of the DES together as solids and at the same time asthe sample. For example, in a single tube, 1.28 g of Choline chloridesolid was added to 2 g of Trifluoroacetamide solid and then 50-100 μl ofwhole blood or 25 mg of tissue sample added and the solids allowed tofreely mix and form a eutectic mixture of (1:2 mol:mol) in the presenceof the biological sample. Alternatively, the two solids can be added astwo preloaded layers in a suitable vessel such as a blood collectiontube, but separated by a membrane which ruptures or dissolves on contactwith the sample thereby allowing the components to mix and form the DESliquid only in the presence of sample. Another possibility is to havetwo open compartments in a suitably closed top vessel each compartmentbeing preloaded with an appropriate amount of, for example, Cholinechloride and the other Trifluoroacetamide. On shaking or inversion thetwo components can freely mix and form a DES liquid, if needed in thepresence of the sample.

36. RNA Stabilisation with Adherent Tissue Culture Cells

Human Embryonic Fibroblast cells (HEF) were grown to 80% confluence(approximately 200,000 cells) in a 24-well tissue culture plate, thegrowth medium removed and replaced with 400 μl of either Cholinechloride:Trifluoroacetamide (1:2 mol:mol) or RNAlater and incubated at37° C. for 0, 32 hours or 9 days prior to RNA purification and RINanalysis (Agilent Bioanalyser). Table 13 shows that adherent tissueculture cell RNA can be extremely well preserved using either Cholinechloride:Trifluoroacetamide (1:2 mol:mol).

TABLE 13 RIN Scores for RNA extracted from Human Embryonic Fibroblast(HEF) adherent cells stored at 37° C. Treatment Time RIN 1 Control 0 9.12 Choline chloride:Trifluoroacetamide 32 hours 9.1 (1:2 mol:mol) 3Choline chloride:Trifluoroacetamide  9 days 8 (1:2 mol:mol) 4 Control 08.8 5 RNAlater ® 32 hours 9.4 6 RNAlater ®  9 days 7.6

1-159. (canceled)
 160. A composition comprising a eutectic solvent,wherein said eutectic solvent comprises a first component and a secondcomponent; wherein said first component is trimethylglycine and saidsecond component is trifluoroacetamide; wherein said first component andsaid second component are in a molar ratio of between about 1:1.5 andabout 1:2.5; and wherein said first component and said second componenttogether comprise at least about 88% by weight of the composition. 161.The composition of claim 160, which further comprises a samplecomprising one or more of a biomolecule, RNA, DNA, protein,phosphoprotein, virus, cell, tissue, solid tissue, plasma, serum, brain,cerebrospinal fluid, whole blood, or whole blood comprising acirculating tumor cell.
 162. The composition of claim 160, which furthercomprises a sample comprising one or more of viruses, influenza, humanpapillomavirus, human immunodeficiency virus, hepatitis A virus,hepatitis C virus, hepatitis E virus, foot and mouth disease virus,severe acute respiratory syndrome virus, West Nile virus, Ebola virus,yellow fever virus, dengue fever virus, bacteria, Escherichia coli,Staphylococcus spp., Streptococcus spp., Mycobacterium spp., Pseudomonasspp., bacteria that cause shigella, diphtheria, tetanus, syphilis,chlamydia, legionella, listeria, or leprosy, parasites, Leishmania spp.,Trypanosoma spp., or Plasmodium spp.
 163. The composition of claim 161,wherein said eutectic solvent prevents degradation of a biomolecule insaid sample.
 164. The composition of claim 160, wherein the molar ratioof said first component to said second component is about 1:2.
 165. Thecomposition of claim 160, wherein the eutectic solvent has a pH between5 and 7.5.
 166. The composition of claim 160, wherein the eutecticsolvent further comprises zinc sulfate, a desiccant, sodium dodecylsulfate, sodium benzoate, N-methyl pyrrolidone, N-ethyl pyrrolidone,1-methylimidazole, 1-benzylimidazole, tetramethylurea, 4-Formylmorpholine, water, and/or acetic acid.
 167. The composition of claim166, wherein said desiccant comprises a silica gel and/or a molecularsieve.
 168. The composition of claim 166, wherein said zinc sulfate ispresent in the eutectic solvent in an amount in the range 0.01% to 1.5%by weight of the eutectic solvent.
 169. A composition comprising aeutectic solvent, wherein said eutectic solvent comprises a firstcomponent and a second component; wherein said first component istrimethylglycine and said second component is trifluoroacetamide;wherein said first component and said second component are in a molarratio of between about 1:1.8 and about 1:2.2; and wherein said firstcomponent and said second component together comprise at least about 88%by weight of the composition.
 170. The composition of claim 169, whichfurther comprises a sample comprising one or more of a biomolecule, RNA,DNA, protein, phosphoprotein, virus, cell, tissue, solid tissue, plasma,serum, brain, cerebrospinal fluid, whole blood, or whole bloodcomprising a circulating tumor cell.
 171. The composition of claim 169,which further comprises a sample comprising one or more of viruses,influenza, human papillomavirus, human immunodeficiency virus, hepatitisA virus, hepatitis C virus, hepatitis E virus, foot and mouth diseasevirus, severe acute respiratory syndrome virus, West Nile virus, Ebolavirus, yellow fever virus, dengue fever virus, bacteria, Escherichiacoli, Staphylococcus spp., Streptococcus spp., Mycobacterium spp.,Pseudomonas spp., bacteria that cause shigella, diphtheria, tetanus,syphilis, chlamydia, legionella, listeria, or leprosy, parasites,Leishmania spp., Trypanosoma spp., or Plasmodium spp.
 172. Thecomposition of claim 170, wherein said eutectic solvent preventsdegradation of a biomolecule in said sample.
 173. The composition ofclaim 169, wherein the molar ratio of said first component to saidsecond component is 1:2.
 174. The composition of claim 169, wherein theeutectic solvent has a pH between 5 and 7.5.
 175. The composition ofclaim 169, wherein the eutectic solvent further comprises zinc sulfate,a desiccant, sodium dodecyl sulfate, sodium benzoate, N-methylpyrrolidone, N-ethyl pyrrolidone, 1-methylimidazole, 1-benzylimidazole,tetramethylurea, 4-Formyl morpholine, water, and/or acetic acid. 176.The composition of claim 175, wherein said desiccant comprises a silicagel and/or a molecular sieve.
 177. The composition of claim 175, whereinsaid zinc sulfate is present in the eutectic solvent in an amount in therange 0.01% to 1.5% by weight of the eutectic solvent.